CHIMERIC ANTIGEN RECEPTORS (CARs) TARGETING HEMATOLOGIC MALIGNANCIES, COMPOSITIONS AND METHODS OF USE THEREOF

ABSTRACT

The present disclosure provides chimeric antigen receptor polypeptides having antigen recognition domains for CD2, CD3, CD4, CD5, CD7, CD8, and CD52 antigens, and polynucleotides encoding for the same. The present disclosure also provides for engineered cells expressing the polynucleotide or polypeptides. In some embodiments, the disclosure provides methods for treating diseases associated with CD2, CD3, CD4, CD5, CD7, CD8, and CD52 antigens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/551,862, filed on Aug. 17, 2017, which is a national stage filingunder 35 USC § 371 of international application numberPCT/US2016/019953, filed on Feb. 26, 2016, claiming priority from U.S.Provisional Application No. 62/121,842, filed Feb. 27, 2015, all ofwhich are incorporated herein by reference in their entirety.

BACKGROUND

T cells, a type of lymphocyte, play a central role in cell-mediatedimmunity. They are distinguished from other lymphocytes, such as B cellsand natural killer cells (NK cells), by the presence of a T-cellreceptor (TCR) on the cell surface. T helper cells, also called CD4+ Tor CD4 T cells, express CD4 glycoprotein on their surface. Helper Tcells are activated when exposed to peptide antigens presented by MHC(major histocompatibility complex) class II molecules. Once activated,these cells proliferate rapidly and secrete cytokines that regulateimmune response. Cytotoxic T cells, also known as CD8+ T cells or CD8 Tcells, express CD8 glycoprotein on the cell surface. The CD8+ T cellsare activated when exposed to peptide antigens presented by MHC class Imolecules. Memory T cells, a subset of T cells, persist long term andrespond to their cognate antigen, thus providing the immune system with“memory” against past infections and/or tumor cells.

T cells can be genetically engineered to produce special receptors ontheir surface called chimeric antigen receptors (CARs). CARs areproteins that allow the T cells to recognize a specific protein(antigen) on tumor cells. These engineered CAR T cells are then grown inthe laboratory until they number in the billions. The expandedpopulation of CAR T cells is then infused into the patient.

Clinical trials to date have shown chimeric antigen receptor (CAR) Tcells to have great promise in hematologic malignancies resistant tostandard chemotherapies. Most notably, CD19-specific CAR (CD19CAR)T-cell therapies have had remarkable results including long-termremissions in B-cell malignancies (Kochenderfer, Wilson et al. 2010,Kalos, Levine et al. 2011, Porter, Levine et al. 2011, Davila, Riviereet al. 2013, Grupp, Frey et al. 2013, Grupp, Kalos et al. 2013, Kalos,Nazimuddin et al. 2013, Kochenderfer, Dudley et al. 2013, Kochenderfer,Dudley et al. 2013, Lee, Shah et al. 2013, Park, Riviere et al. 2013,Maude, Frey et al. 2014).

Despite the success of CAR therapy in B-cell leukemia and lymphoma, theapplication of CAR therapy to T-cell malignancies has not yet been wellestablished. Given that T-cell malignancies are associated withdramatically poorer outcomes compared to those of B-cell malignancies(Abramson, Feldman et al. 2014), CAR therapy in this respect has thepotential to further address a great clinical need.

CD5 is expressed in more than 80% of T-cell acute lymphoblastic leukemia(T-ALL). One treatment option is to treat patients with anti-CD5antibodies as T-cell leukemias or T-cell lymphomas expressing the CD5surface molecule. However attempts have met limited success.

Therefore, there remains a need for improved chimeric antigenreceptor-based therapies that allow for more effective, safe, andefficient targeting of T-cell associated malignancies.

SUMMARY OF THE INVENTION

The present disclosure provides chimeric antigen receptors (CARS)targeting hematologic malignancies, compositions and methods of usethereof.

In one embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD2 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD3 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD4 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD5 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD7 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD8 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polypeptide, the polypeptide comprising: a signalpeptide, a CD52 antigen recognition domain, a hinge region, atransmembrane domain, at least one co-stimulatory domain, and asignaling domain.

In one embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD2.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD3.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD4.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD5.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD7.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD8.

In another embodiment, the disclosure provides an engineered chimericantigen receptor polynucleotide that encodes for a chimeric antigenreceptor polypeptide having an antigen recognition domain selective forCD52.

In one embodiment, the disclosure provides an engineered cell expressingany of the chimeric antigen receptor polypeptides described above.

In another embodiment, the disclosure provides an engineered cellexpressing any of the chimeric antigen receptor polynucleotidesdescribed above.

In another embodiment, the disclosure provides a method of producing anengineered cell expressing a chimeric antigen receptor polypeptide orpolynucleotide having an antigen recognition domain selective for CD2,CD3, CD4, CD5, CD7, CD8, or CD52. The method includes (i) providingperipheral blood cells or cord blood cells; (ii) introducing theaforementioned polynucleotide into the aforementioned cells; (iii)expanding the cells of step (ii); and isolating the cells of step (iii)to provide said engineered cell.

In another embodiment, the disclosure provides a method of producing anengineered cell expressing a chimeric antigen polypeptide orpolynucleotide having an antigen recognition domain selective for CD2,CD3, CD4, CD5, CD7, CD8, or CD52. The method includes (i) providingplacental cells, embryonic stem cells, induced pluripotent stem cells,or hematopoietic stem cells; (ii) introducing the aforementionedpolynucleotide into the cells of step (i); (iii) expanding the cells ofstep (ii); and (iv) isolating the cells of step (iii) to provide saidengineered cell.

In one embodiment, the disclosure provides a method of conferringanti-leukemia or anti lymphoma immunity to CD4 positive T-cell leukemiaor CD4 positive T-cell lymphoma in a patient in need thereof. The methodincludes (i) administering to a patient in need thereof atherapeutically effective amount of an engineered cell expressing a CARpolypeptide having a CD4 antigen recognition domain; and (ii)optionally, assaying for immunity to T-cell leukemia or T-cell lymphomain the patient.

In another embodiment, the disclosure provides a method of reducing thenumber of CD4 positive T-cell leukemia cells or CD4 positive T-celllymphoma cells. The method includes (i) contacting CD4 positive T-cellleukemia cells or CD4 positive T-cell lymphoma cells with an effectiveamount of an engineered cell expressing a CAR polypeptide having a CD4antigen recognition domain; and (ii) optionally, assaying for CD4positive T-cell leukemia cells or CD4 positive T-cell lymphoma cells.

In another embodiment, the disclosure provides a method of reducing thenumber of immunoregulatory cells having a CD2 antigen. The methodincludes (i) contacting said immunoregulatory cells with an effectiveamount of an engineered cell expressing a CAR polypeptide having a CD2antigen recognition domain; and (ii) optionally, assaying for thereduction in the number of immunoregulatory cells.

In another embodiment, the disclosure provides a method of reducing thenumber of immunoregulatory cells having CD3. The method includes (i)contacting said immunoregulatory cells with an effective amount of anengineered cell expressing a CAR polypeptide having a CD3 antigenrecognition domain; and (ii) optionally, assaying for the reduction inthe number of immunoregulatory cells.

In another embodiment, the disclosure provides a method of reducing thenumber of immunoregulatory cells having CD4. The method includes (i)contacting said immunoregulatory cells with an effective amount of anengineered cell expressing a CAR polypeptide having a CD4 antigenrecognition domain; and (ii) optionally, assaying for the reduction inthe number of immunoregulatory cells.

In another embodiment, the disclosure provides a method of reducing thenumber of immunoregulatory cells having CD5. The method includes (i)contacting said immunoregulatory cells with an effective amount of anengineered cell expressing a CAR polypeptide having a CD5 antigenrecognition domain; and (ii) optionally, assaying for the reduction inthe number of immunoregulatory cells.

In another embodiment, the disclosure provides a method of reducing thenumber of immunoregulatory cells having CD7. The method includes (i)contacting said immunoregulatory cells with an effective amount of anengineered cell expressing a CAR polypeptide having a CD7 antigenrecognition domain; and (ii) optionally, assaying for the reduction inthe number of immunoregulatory cells.

In another embodiment, the disclosure provides method of reducing thenumber of immunoregulatory cells having a CD8 antigen. The methodincludes (i) contacting said immunoregulatory cells with an effectiveamount of an engineered cell expressing a CAR polypeptide having a CD8antigen recognition domain; and (ii) optionally, assaying for thereduction in the number of immunoregulatory cells.

In another embodiment, the disclosure provides a method of reducing thenumber of immunoregulatory cells having CD52. The method includes (i)contacting said immunoregulatory cells with an effective amount of anengineered cell expressing a CAR polypeptide having a CD52 antigenrecognition domain; and (ii) optionally, assaying for the reduction inthe number of immunoregulatory cells.

In one embodiment, the disclosure provides a method of treating a cellproliferative disease. The method includes (i) administering to apatient in need thereof a therapeutically effective amount of anengineered cell expressing a CAR polypeptide having a CD2, CD3, CD4,CD5, CD7, CD8, or CD52 antigen recognition domain.

In one embodiment, the disclosure provides a method of treating anautoimmune disease. The method includes (i) administering to a patientin need thereof a therapeutically effective amount of an engineered cellexpressing a CAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8, orCD52 antigen recognition domain.

In one embodiment, the disclosure provides engineered cells expressing aCAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8, or CD52 antigenrecognition domain for use in the treatment of a cell proliferativedisease. The use includes administering said engineered cells to apatient in need thereof.

In some embodiments, CARs typically include at least one ofintracellular signaling, hinge and/or transmembrane domains.First-generation CARs include CD3z as an intracellular signaling domain,whereas second-generation CARs include a single co-stimulatory domainderived from, for example, without limitation, CD28 or 4-1BB. Thirdgeneration CARs include two co-stimulatory domains, such as, withoutlimitation, CD28, 4-1BB (also known CD137) and OX-40, and any otherco-stimulatory molecules.

In some embodiments, CAR having a CD2, CD3, CD4, CD5, CD7, CD8, or CD52antigen recognition domain is part of an expression cassette. In apreferred embodiment, the expressing gene or the cassette may include anaccessory gene or a tag or a part thereof. The accessory gene may be aninducible suicide gene or a part thereof, including, but not limited to,caspase 9 gene. The “suicide gene” ablation approach improves safety ofthe gene therapy and kills cells only when activated by a specificcompound or a molecule. In some embodiments, the epitope tag is a c-myctag, streptavidin-binding peptide (SBP), truncated EGFR gene (EGFRt) ora part or a combination thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1C. CD4CAR expression. (FIG. 1A), Schematic representation ofrecombinant lentiviral vectors encoding CD4CAR. CD4CAR expression isdriven by a SFFV (spleen focus-forming virus) promoter. The thirdgeneration of CD4 CAR contains a leader sequence, the anti-CD4scFv, ahinge domain (H), a transmembrane domain (TM) and intracellularsignaling domains as follows: CD28, 4-1BB (both co-stimulators), and CD3zeta. (FIG. 1B), 293FT cells were transfected with lentiviral plasmidsfor GFP (lane 1) and CD4CAR (lane 2) for Western blot analysis at 48 hpost transfection and probed with mouse anti-human CD3z antibody. (FIG.1C), Illustration of the components of third-generation chimeric antigenreceptor T cells targeting CD4 expressing cells.

FIGS. 2A-2D. Production of CD4CAR T cells. (FIG. 2A), experimentaldesign. (FIG. 2B), CB buffy coat cells were activated 2 days withanti-CD3 antibody and IL-2. Cells were transduced with either GFP(middle) or CD4CAR (right) lentiviral supernatant. After 7 days ofincubation, cells were analyzed by flow cytometry with goat anti-mouseFab2 or goat IgG antibodies conjugated with biotin and followed bystreptavidin-PE. Non-transduced, labeled CB cells are shown on the left.(FIG. 2C), CD4CAR T cells deplete the CD4+ population during T cellexpansion. CB buffy coat cells were activated for 2 days with anti-CD3antibody and IL-2. CB buffy coat contains two subsets of T cells, CD8+cytotoxic T cells and CD4+ helper T cells (left). Cells were transducedwith either GFP (middle) or CD4CAR (right) lentiviral supernatant. After3 day culture, cells were analyzed by flow cytometry withmouse-anti-human CD4 (FITC) and CD8 (APC) antibodies. Non-transducedPMBCs were also labeled (left). (FIG. 2D), Most CD4CAR T cells have acentral memory-like phenotype. CB buffy coat cells were activated 2 dayswith anti-CD3 antibody. Cells were transduced with CD4CAR lentiviralsupernatant. After 6 day expansion, CD8+ cells were analyzed for CD62L,CD45RO and CD45RA phenotypes by flow cytometry (N=3).

FIGS. 3A-3D. CD4CAR T cells eliminate T-cell leukemic cells inco-culture assays. (FIG. 3A), CD4CAR T cells eliminate KARPAS 299 T-cellleukemic cells in co-culture. Activated human CB buffy coat cellstransduced with either GFP (middle) or CD4CAR (right) lentiviralsupernatant were incubated with KARPAS 299 cells at a ratio of 2:1.After 24 hours co-culture, cells were stained with mouse-anti-human CD4(APC) and CD8 (PerCp) antibodies and analyzed by flow cytometry for Tcell subsets (N=3). (FIG. 3B) and (FIG. 3C), CD4CAR T cells eliminateprimary T-cell leukemic cells in co-culture. Activated human CB buffycoat cells transduced with either GFP (middle) or CD4CAR (right)lentiviral supernatant were incubated with primary T-cell leukemia cellsfrom Sezary syndrome (FIG. 3B) and PTCLs (FIG. 3C) at a ratio of 2:1.After 24 hours of co-culture, cells were analyzed by flow cytometry withmouse-anti-human CD4 (FITC) and CD8 (APC) antibodies (N=3). Humanprimary cells alone are also labeled (left). (FIG. 3D) CD4CAR T cellswere unable to lyse CD4-negative lymphoma cells (SP53, a B-cell lymphomacell line). Activated human CB buffy coat cells transduced with eitherGFP (middle) or CD4CAR (right) lentiviral supernatant were incubatedwith SP53 mantle cell lymphoma cells which were pre-stained with themembrane dye CMTMR, at a ratio of 2:1. After 24 hours co-culture, cellswere stained with mouse-anti-human CD3 (PerCp) and then analyzed by flowcytometry (N=2). SP53 cells alone, pre-stained with CMTMR were alsolabeled (left).

FIGS. 4A-4B. CD4CAR T cells derived from PBMCs are highly enriched forCD8+ T and specifically kill CD4-expressing leukemic cell lines. (FIG.4A) CD4CAR T cells derived from PBMCs are highly enriched for CD8+ Tcells. PMBC buffy coat cells constituting T cells, CD8+ and CD4+ (left)were activated for 2 days with anti-CD3 antibody and IL-2, thentransduced with either GFP (middle) or CD4CAR (right) lentiviralsupernatant. After 3 days of culture, cells were labeled and analyzed byflow cytometry for T cell subsets. Non-transduced PMBCs were alsolabeled (left). (FIG. 4B) CD4CAR T cells specifically kill KARPAS 299cells. PMBC T cells transduced with either GFP control or CD4CARlentiviral supernatant were incubated with CFSE-stained KARPAS 299 atthe ratios of 2:1, 5:1 and 10:1, respectively. After overnightincubation at 37° C., dye 7AAD was added, and the cells were analyzed byflow cytometry. Percent killing of target cells is measured by comparingsurvival of target cells relative to the survival of negative controlcells (SP53 cells, a B-cell lymphoma cell line stained with CMTMR).

FIGS. 5A-5D. CD4CAR T cells efficiently mediate anti-leukemic effects invivo with different modes. NSG mice received 2.5 Gy for sub-lethalirradiation. Twenty-four hours after irradiation, mice were injectedsubcutaneously with either 1×10⁶ (in A) or 0.5×10⁶ (in FIGS. 5B and 5C)KARPAS 299 cells. Injected mice were treated with different courses andschedules of CD4CAR T cells or control T cells. N=5 for each group ofinjected mice. (FIG. 5A), a low dose of 2×10⁶ of CD4CAR T cells wasinjected on day 3 followed by a large dose, 8×10⁶, of CD4CAR T cells onday 22 after upon observed acceleration of tumor growth. (FIG. 5B), twolarge doses of CD4CAR T cells, 8×10⁶ and 5.5×10⁶ were injected on day 3and 10 respectively. (FIG. 5C), a repeat low dose (2.5×10⁶) of CD4CAR Tcells was injected every 5 days for a total of four administrations.(FIG. 5D), overall survival of mice treated with the indicated CD4CAR Tcells or control GFP T cells. N=10.

FIG. 6. CD4CAR is expressed on the surface in HEK-293 cells. HEK-293cells were transduced for 6 hours with CD4CAR or GFP control viralsupernatant. Following a 3 day incubation, cells were analyzed by flowcytometry.

FIG. 7. Comparison of cell growth between activated PMBC buffy coatcells transduced with lenti-GFP and CD4CAR viruses. The activated PMBCbuffy coat cells were transduced with either GFP control or CD4-CARlentiviral supernatant on Day 0. Cells were washed on Day 1, and mediawas added on days 3 and 5.

FIGS. 8A-8C. CD4CAR construct. (FIG. 8A) Schematic representation oflentiviral vector encoding third generation CD4CAR, driven by spleenfocus-forming virus (SFFV) promoter. The construct contains a leadersequence, anti-CD4 scFv, hinge domain (H), transmembrane (TM) andsignaling domains CD28, 4-1BB, and CD3 zeta. (FIG. 8B) HEK293FT cellswere transfected with GFP vector control (lane 1) and CD4CAR (lane 2)lentiviral plasmids. Forty-eight hours after transfection, cells wereremoved and subsequently used for Western blot analysis with mouseanti-human CD3z antibody. (FIG. 8C) Illustration of third-generation CARNK cells targeting CD4 expressing cells.

FIG. 9. CD4CAR NK cell production. (A, upper panel) CD4CAR expressionlevels on NK cells prior to being sorted by FACS (N=3); (A, lower panel)CD4CAR expression on NK cells after sorting and expansion, prior toco-culture experiments (N=3)

FIGS. 10A-10C. CD4 CAR NK cells ablate CD4⁺ leukemia and lymphoma cellsin co-culture assays. Co-culture experiments were performed at aneffector to target ratio of 2:1 for 24 hours and were directly analyzedby flow cytometry for CD56 and CD4 (panels FIGS. 10A and 10B). Eachassay consists of target cells alone control (left), and target cellsincubated with NK cells transduced with vector control (center) orCD4CAR (right) lentiviral supernatant. Top row, panel A: Karpas 299(N=3). Middle row, panel A: HL-60 T-cells (N=2). Bottom row, panel A:CCRF-CEM cells (N=2). CD4CAR NK cells eliminated primary T-cell leukemiacells from a patient with CD4⁺ T-cell lymphoma Sézary syndrome (N=2) andCD4 expressing pediatric T-cell ALL (N=2). (FIG. 10C) Bar graphsummarizing co-culture assay results for both 2:1 and 5:1 E:T ratios.

FIG. 11. Co-culture specificity and dose response killing curve. CD4CARNK cells lyse CD4-expressing leukemic cell lines in a dose dependent andspecific manner. CD4CAR NK and vector control cells were incubated withan equal ratio of CFSE-stained “on-target” (Karpas 299 or CCRF-CEM)cells and CMTMR-stained “off target” MOLT4 cells at 1:4, 1:2, and 1:1effector to target ratios. After 24 hours, 7-AAD dye was added andremaining live cells were analyzed by flow cytometry. Percent killing oftarget cells was measured by comparing CD4⁺ Karpas 299 or CCRF-CEM cellsurvival in CD4CAR NK cell co-cultures relative to that in vectorcontrol NK cell co-cultures.

FIGS. 12A-12B. CD4CAR NK cells eliminate CD4⁺ T-cells isolated fromhuman cord blood at an effector to target ratio of 2:1, but do notaffect hematopoietic stem cell/progenitor compartment output. (FIG. 12A)Co-culture assays were performed at an effector to target ratio of 2:1for 24 hours, after which, cells were stained with mouse anti-human CD56and CD4 antibodies. Target cells were incubated alone as a control(left). NK cells were transduced with either vector control (center) orCD4CAR (right) lentiviral supernatant and incubated with CD4⁺ T-cellsobtained from human cord blood. (N=2) (FIG. 12B) CD4CAR NK cells wereincubated at co-culture effector:target ratios of 2:1 and 5:1respectively with 500 CD34+ cord blood cells for 24 hours in NK cellmedia supplemented with IL-2. Experimental controls used were CD34+cells alone, and non-transduced NK cells were co-cultured at respective2:1 and 5:1 effector:target ratios with CD34+ CB cells. Hematopoieticcompartment output was assessed via formation of erythroid burst-formingunits (BFU-E) and number of granulocyte/monocyte colony-forming units(CFU-GM) at Day 16. CFU statistical analysis was performed via 2-wayANOVA with alpha set at 0.05.

FIGS. 13A-13D. CD4CAR NK cells demonstrate anti-leukemic effects invivo. NSG mice were sublethally irradiated and intradermally injectedwith luciferase-expressing Karpas 299 cells (Day 0) to induce measurabletumor formation. On day 1 and every 5 days for a total of 6 courses,mice were intravenously injected with 5×10⁶ CD4CAR NK cells or vectorcontrol NK control cells. (FIG. 13A) On days 7, 14, and 21, mice wereinjected subcutaneously with RediJect D-Luciferin and subjected to IVISimaging. (FIG. 13B) Average light intensity measured for the CD4CAR NKinjected mice was compared to that of vector control NK injected mice.(FIG. 13C) On day 1, and every other day after, tumor size area wasmeasured and the average tumor size between the two groups was compared.(FIG. 13D) Percent survival of mice was measured and compared betweenthe two groups.

FIG. 14. CD4 CAR NK cells ablate CD4 positive leukemia and lymphomacells in co-culture assays. All co-culture assays shown were performedat an effector to target ratio of 5:1 for 24 hours, after which, cellswere stained with mouse anti-human CD56 and CD4 antibodies. Each assayconsists of NK cells transduced with either vector control (center) orCD4CAR (right) lentiviral supernatant and incubated with target cells,as well as target cells incubated alone as a control (left). CD4CAR NKcells eliminated Karpas 299 leukemic T-cells (A), HL-60 T-cells (B), andCCRF-CEM cells (C). CD4CAR NK cells eliminated primary T-cell leukemiacells from patients with CD4 expressing T-cell leukemia/Sézary syndrome(D) and CD4 expressing pediatric T-cell ALL (E).

FIG. 15. NK cells were transduced with either vector control or CDCARlentiviral supernatant, or cultured for non-transduced control. After 7days of incubation, cells were harvested and analyzed by flow cytometrywith Biotin-labeled goat anti-mouse F(Ab′)2 followed by streptavidin-PE.NK cells were >85% CD4CAR⁺ after sorting.

FIGS. 16-16C. CD4CAR NK cells did not lyse CD4⁻, CD5⁺ MOLT4 negativecontrol. (FIG. 16A) MOLT4 cell immunophenotype was confirmed to bealmost all CD4⁻ and CD5⁺. (FIG. 16B) CD4CAR NK cells did not lyse MOLT4cells at a 5:1 effector to target ratio at 0 h, 4 h, 8 h, and 24 h(lower panel) as assessed by comparison to vector control NK celltumorlysis (upper panel). (FIG. 16C) Anti-CD4 CDCAR NK antitumoractivity was confirmed at 4 h with a CD4⁺ Karpas 299 positive control atan 5:1 E:T ratio.

FIGS. 17A-17C. Generation of CD5CAR. FIG. 17A. The DNA gene constructand the translated protein construct for CD5CAR, and anchored CD5 scFvantibody and a cartoon demonstrating the creation and function ofCD5CAR. The DNA construct of the third generation CD5CAR construct from5′ to 3′ reads: Leader sequence, the anti-CD5 extracellular single chainvariable fragment (Anti-CD5 ScFv), the hinge region, the trans-membraneregion, and the three intracellular signaling domains that define thisconstruct as a 3rd generation car; CD28, 4-1BB and CD3ζ. The DNAconstruct of the anchored CD5 scFv antibody is the same as the CD5CARconstruct without the intracellular signaling domains, as is thetranslated protein product for anchored CD5 scFv antibody. Thetranslated protein constructs contain the anti-CD5 ScFv that will bindto the CD5 target, the hinge region that allows for appropriatepositioning of the anti-CD5 ScFv to allow for optimal binding position,and the trans-membrane region. The complete CD5CAR protein also containsthe two co-stimulatory domains and an intracellular domain of CD3 zetachain. This construct is considered as a 3rd generation CAR: CD28,4-1BB, and CD3ζ. FIG. 17B. Western blot analysis demonstrates the CD5CARexpression in HEK293 cells. HEK293 cells which had been transduced withGFP (as negative control) or CD5CAR lentiviruses for 48 h were used forWestern blot analysis using CD3ζ antibody to determine the expression ofCD5CAR. Left lane, the GFP control HEK293 cells, with no band asexpected. The right lane showing a band at about 50 kDa, the molecularweight that we expected based on the CD5CAR construct. FIG. 17C. Flowcytometry analysis for CD5CAR expression on T cells surface forlentiviral transduced CD5CAR T cells. This analysis was performed on thedouble transduced CD5CAR T cells at day 8 after the second lentiviraltransduction. Left: isotype control T cell population (negativecontrol); right, transduced T cells expressing CD5 CAR showing 20.53% onT cells by flow cytometry using goat anti-mouse F(AB′)2-PE.

FIGS. 18A-18C. Study Schema of the transduction of CD5CAR T-cells. FIG.18A. Steps for generation of CD5 CAR T cells by single transduction.FIG. 18B. Steps for generation of CD5 CAR T cells by doubletransduction. FIG. 18C. Comparisons of single and double transductionswith CD5 CAR lentviruses in the down-regulation of surface CD5expression on the T cells. The down-regulation of extracellular CD5protein versus GFP T-cell control over 8 days following lentiviraltransduction is analyzed. The single transduced CD5CAR T-cells do notshow complete downregulation of CD5 from cell surface by day 8, with amaximum decrease in CD5 protein expression on day 6. In the doubletransduced population, we note the decrease in the absolute number ofCD5+, CD3+ double positive CD5CAR T-cells over time, from 24.44% on day0 to a near complete reduction of CD5 expression on day 4. In contrast,the GFP T-cell control maintains a CD5+, CD3+ double positive populationabove 95% from day 2 through day 8.

FIGS. 19A-19B. Downregulation of CD5 expression on T-cells afterlentiviral transduction of anchored CD5 scFv antibody after 7 days. FIG.19A. Study schema for the transduction of anchored CD5 scFvlentiviruses, single transduction. FIG. 19B. Anchored CD5 scFvdown-regulates or reduces the quantity of surface CD5 expression on Tcells. Flow cytometry analysis demonstrating the significant decrease inCD5 protein expression (˜32%) after single transduction of CD5 scFv and7 day incubation. Elimination of CD5 expression is observed, but notcomplete after 7 days, and a follow up study is currently beingcompleted for a double transduced anchored CD5 scFv antibody.

FIGS. 20A-20B. CD5CAR cells effectively lyse T-ALL cell lines thatexpress CD5, and do not lyse a T leukemic cell line that does notexpress CD5. FIG. 20A. Flow cytometry analysis of T-ALL cell lines alone(left column), in co-culture with GFP vector transduced T-cells (middlerow) and in co-culture with CD5CAR transduced T-cells (right row). Eachcell line is seen in each row, The CD5+ T-ALL cell lines in the top andmiddle rows (CCRF-CEM and Molt-4) with the CD5 negative cell line seenas the bottom row (KARPAS 299). KAEPAS 299 is a CD5 negative T celllymphoma. The incubation time for all co-cultures was 24 hrs, with aneffector:target cell ratio of 5:1. The cell lysis compared to GFPcontrol was over 78% for both CD5 T ALL leukemic cell lines, compared tothat for the GFP control. FIG. 20B. This bar graph denotes the T celllysis achieved by the CD5CAR T-cells when compared to the GFP T-cellsco-culture described in FIG. 20A. There was no lysis observed in CD5 CART cells co-cultures with KARPAS 299, which is CD5 negative (n=3independent experiments done in duplicate).

FIGS. 21A-21D. CD5CAR cells effectively lyse T-cell acute lymphoblasticleukemic cells from patient samples that express CD5. FIG. 21A. Flowcytometry analysis of T-ALL cells alone (left column), in co-culturewith GFP T-cells (middle row) and in co-culture with CD5CAR T-cells(right row). Each patient cells are given a row, and are numbered tomaintain patient confidentiality. The incubation time for allco-cultures was 24 hrs, with an effector:target cell ratio of 5:1. Thecell lysis compared to GFP control was over 71.3% for the T-ALL-1compared to control. The rest of the cell lines demonstrated positivecell lysis as well, but to a lesser degree, between 33-47%. This may berelated to the CD5 expression for each leukemic sample, which isdiscussed below. FIG. 21B. This bar graph denotes the T cell lysisachieved by the CD5CAR T-cells when compared to the GFP T-cellco-culture described in FIG. 21A. All experiments were done induplicate. FIG. 21C. Flow cytometry analysis data demonstrating CD3 andCD5 expression levels for patient T cell ALL samples analyzed in FIG.21A. We observe a different CD5 positivity for T-ALL 1 and T-ALL 3. D.Flow cytometry analysis of the levels of CD5 expression on a panel offour patient sample T-ALL cell populations. The difference of meanfluorescent intensity (MFI) was determined by flow cytometry analysis(FIG. 21C).

FIG. 22. Analysis of CD5CAR T-cell killing ability for patient T-ALLcells (T-ALL-8) in details. Flow cytometry analysis demonstrating CD5CART-cell killing ability for patient's T-ALL cells. The control GFP-T celland T-ALL-8 cell co-culture are seen on the left, and the CD5CARco-culture with T-ALL 8 is seen on the right. We note avid lysis of allCD5 positive cells, both CD34 positive (circled in red) and CD34negative (circled in green, T cells), with no lysis noted for CD5negative cells. When compared to GFP control, CD5CAR T cells lyse atminimum 93.1% of CD5 positive T-ALL-8 cells when compared to GFPcontrol. Experiment was done in duplicate. In addition, CD5CAR T cellsessentially eliminate the T cell population (CD5+CD34−, circled ingreen).

FIGS. 23A-23B. CD5CAR T cells effectively eliminate normal GFP labeled Tcells.

FIG. 23A, CD5CAR T cells kill normal T cells in a dose dependent manner.CD5CAR T cells or CD123CAR T cells (control) were co-cultured with GFPlabeled T cells at 0.25:1, 0.5:1 and 1:1 effector to target ratios.After 24 hours, remaining live GFP T cells were analyzed by flowcytometry. Percent killing of target cells was measured by comparing GFPT cell survival in CD5 co-cultures relative to that in control CD123CART cells as T cells do not express CD123. FIG. 23B, Co-culture killingcurve based on the data from A.

FIGS. 24A-24B. T cells maintained CD5 expression when they wereco-cultured with CD5CAR or anchored CD5 scFv T cells. FIG. 24A, Stepsfor generation of CD5CAR T cells or anchored CD5 scFv T cells and CD123CAR T cells (control). FIG. 24B, CD5 expression levels on different CARtransduced T-cells (Day 3 after 2^(nd) transduction). Activated T cellswere transduced with lentiviruses expressing CD5CAR or anchored CD5 scFvand CD123CAR. After 3 day transduction, CD5 expression was analyzed byflow cytometry.

FIGS. 25A-25C. Co-culture assays were performed to determine if normal Tcells maintained CD5 expression when they were co-cultured with CD5CARor anchored CD5 scFv T cells or CD123CAR (control) for 2 days (FIG. 25A)or 4 days (FIG. 25B) at a ratio of 1:1. CD5CAR T cells or anchored CD5scFv T cells or CD123CAR T (control) cells were incubated with GFPlabeled T cells and the co-cultured GFP labeled T cells were thenanalyzed for CD5 expression and live cells by flow cytometry. C (FIG.25C), CD5CAR− or anchored CD5 scFv transduced CCRF-CEM or Molt-4 T ALLcells showed downregulation of CD5 expression. CCRF-CEM or Molt-4 T ALLcells were transduced with lentiviruses expressing CD5CAR or anchoredCD5 scFv. After the second transduction, the transduced leukemic cellswere analyzed for CD5 expression by flow cytometry.

FIGS. 26A-26D. CD5CAR T cells demonstrate profound anti-leukemic effectsin vivo. NSG mice were sublethally irradiated and, after 24 hours,intravenously injected with 1×10⁶ luciferase-expressing CCRF-CEM cells(Day 0) to induce measurable tumor formation. On day 3 and 4, mice wereintravenously injected with 5×10⁶ CD5CAR T cells or vector control Tcells. These injections were repeated on Days 6 and 7, for a total of2.0×10⁷ cells per mouse. FIG. 26A, On days 5, 8, 10 and 13, mice wereinjected subcutaneously with RediJect D-Luciferin and subjected to IVISimaging. FIG. 26B, Average light intensity measured for the CD5CAR Tinjected mice was compared to that of vector control T injected mice.FIG. 26C, Percentage of tumor cells killed in mice treated with CD5CAR Tcells relative to control. FIG. 26D, Peripheral blood was drawn frommice on Day 15 and percentages of leukemic cells were determined andcompared to that of vector control or normal injected mice.

FIGS. 27A-27C. The CD5 CAR NK cells (NK-92) effectively eliminateCCRF-CEM T-ALL cell line in vitro. FIGS. 27A and 27B, T-lymphoblast cellline CCRF-CEM expressing CD5 was co-cultured with CD5 CAR NK cells inthe indicated E:T (effector:target) cell ratios for 24 hours. Targetpopulations were quantified with flow cytometry using CD56 and CD5 toseparate the NK-CAR and target cell population respectively. Cellsurvival is expressed relative to transduced vector control NK cells andeach bar graph represents the average statistics for duplicate sampleswith N=2. FIG. 27C, CD5CAR NK cells eliminate CCRF-CEM cells in adose-dependent manner. T-lymphoblast cell line, CCRF-CEM expressing CD5was co-cultured with CD5CAR NK cells in the indicated E:T(effector:target) cell ratios with the lower bound of the E:T ratioreduced. Saturation is achieved with an E:T ratio of 2:1 andco-culturing under reduced ratios results in a dosage-dependent mannerof CD5 elimination. Complete elimination of CCRF-CEM was achieved at5:1.

FIGS. 28A-28B. CD5CAR NK cells effectively lyse two CD5+T-ALL lines,MOLT-4 and Jurkat. FIG. 28A, CD5CAR NK cells were co-cultured withMOLT-4 cells in the indicated E:T (effector:target) cell ratios for 24hours. Cell survival is expressed relative to transduced vector controlNK cells and each bar graph represents the average statistics forduplicate samples with N=2 experiments. FIG. 28B, CD5CAR NK cells areco-cultured with Jurkat cells in the indicated E:T (effector:target)cell ratios for 24 hours. Cell survival is expressed relative totransduced vector control NK cells and each bar graph represents theaverage statistics for duplicate samples with N=2 experiments.

FIGS. 29A-29D. CD5CAR NK cells effectively eliminate aggressive CD5+T-ALL cells using human samples. FIG. 29A, T-ALL cells from patient,T-ALL #1 were co-cultured with CD5CAR NK cells in the indicated E:T(effector:target) cell ratios for 24 hours. FIG. 29B, T-ALL cells frompatient, T-ALL #2 were co-cultured with CD5CAR NK cells in the indicatedE:T (effector:target) cell ratios for 24 hours. Target populations weregated and quantified with flow cytometry using cell cytotracker dye(CMTMR) to screen T-ALL patient samples. Data represents the averagestatistics for duplicate samples. Target CD5+CD34+ cell populations weregated against an isotype control. Cell survival is expressed relative totransduced vector control NK cells and each bar graph. From left toright, the bar graph shows the data for CD34+ CD5+ on the left andCD5+cd34− on the right, for each ratio. CD5CAR NK shows almost completelysis of the highly expressing CD5+ target population with activityagainst the low CD5+CD34+ potential tumor stem cell population.Saturation is achieved at a ratio of 2:1, signifying a need for dilutionof E:T ratios. FIGS. 29C and 29D, leukemic cells from patient #3 (PTCLs)and patient #4 (Sezary Syndrome) were co-cultured with CD5CAR NK cells,respectively in the indicated E:T (effector:target) cell ratios for 24hours.

FIG. 30. CD5NK-CAR specifically eliminates umbilical cord blood T-cells.T cells are isolated from umbilical cord blood (UCB) T-cells andco-cultured with CD5CAR NK cells in the indicated E:T (effector:target)cell ratios for 24 hours. Target populations were quantified with flowcytometry using CD56 and CD5 to separate the NK-CAR and T-cellpopulation respectively. Cell survival is expressed relative totransduced vector control NK cells and each bar graph represents theaverage statistics for duplicate samples.

FIGS. 31A-31C. CD5CAR NK cells effectively eliminate CD5+ mantle celllymphoma and chronic lymphocytic leukemia. CD5CAR NK cells wereco-cultured with Jeko cells (FIG. 31A) and leukemic cells from patientswith mantle cell lymphoma (FIG. 31B) and chronic lymphocytic leukemia(FIG. 31C). Mantle cell line lymphoma derived cell line JeKo expressinga major subset of CD5 was co-cultured with CD5CAR NK cells in theindicated E:T (effector:target) cell ratios for 6 hours. For mantle celllymphoma or CLL, co-cultures were conducted for 24 hours. Targetpopulations were gated and quantified with flow cytometry as illustratedin figures. CD5CAR NK cells specifically targets the CD5+CD19+ leukemiapopulation and the CD5+CD19− T-cell population. Cell survival isexpressed relative to transduced vector control NK cells and each bargraph represents the average statistics for duplicate samples.

FIG. 32. Bars graph summarizing the CD5CAR NK cell co-cultures studies.

FIGS. 33A-33B. CD5CAR NK cells demonstrate potent anti-leukemic effectsin vivo. NSG mice were sublethally irradiated and, after 24 hours,intravenously injected with 1×10⁶ luciferase-expressing CCRF-CEM cells(Day 0) to induce measurable tumor formation. On day 3 and 4, mice wereintravenously injected with 5×10⁶ CD5CAR NK cells or vector control NKcells. These injections were repeated on Days 6 and 7, for a total of2.0×10⁷ cells per mouse. FIG. 33A, on day 5, mice were injectedsubcutaneously with RediJect D-Luciferin and subjected to IVIS imaging.FIG. 33B, Percentage of tumor cells killed in mice treated with CD5CARNK cells relative to control.

FIGS. 34A-34B, Organization of CD3CAR and its expression. FIG. 34A,Schematic representation of the organization of CD3CAR in lentiviralvectors. CAR expression is driven by a SFFV (spleen focus-forming virus)promoter and as a 3^(rd) generation construct, contains a leadersequence, the anti-CD3scFv, a hinge domain (H), a transmembrane domain(TM), two co-stimulatory domains of CD28 and 4-BB and the intracellularsignaling domain of CD3 zeta. FIG. 34B, HEK-293FT cells were transducedwith lentiviral plasmids for GFP (lane 1) and CD3CAR (lane 2) forWestern blot analysis at 48 h post transduction and probed with mouseanti-human CD3zeta antibody.

FIGS. 35A-35B. CD3CAR NK cells eliminate CD3-expressing T-ALL cell linesin vitro. FIG. 35A, T-lymphoblast cell line Jurkat expressingapproximately 80% CD3 was co-cultured with CD3CAR NK cells in theindicated E:T (effector:target) cell ratios for 6 hours. FIG. 35B,Sorted (CCRF-CD3) or unsorted CCRF-CEM (CCRF-CEM) cells were co-culturedwith CD3CAR NK cells for 24 hours. Target populations were quantifiedwith flow cytometry using CD56 and CD3 to separate the NK-CAR and targetcell population respectively. Cell survival is expressed relative totransduced vector control NK cells and each bar graph represents theaverage statistics for duplicate samples with N=2 experiments.

FIGS. 36A-B. The CD3CAR NK cells display robust killing ability forprimary CD3+ leukemic cells from patient samples. FIG. 36A, SPT-1(Sezary syndrome) patient cells were CD3 positive and were co-culturedwith CD3CAR NK cells in the indicated E:T (effector:target) cell ratiosfor 24 hours. Target populations were quantified with flow cytometryusing CD56 and CD3 to separate the NK-CAR and target cell populationrespectively. While SPT-1 is a heterogenous cell population, the broadCD3+ expressing population is eliminated by the CD3NK-CAR. FIG. 36B, PT4(unclassified PTCLs) patient cells were CD3+CD7−, and were co-culturedwith CD3CAR NK cells in the indicated E:T (effector:target) cell ratiosfor 24 hours. Target populations were gated and quantified as seen inthe figure. PT4 leukemia cells are typed CD3+CD7− and are effectivelyeliminated by the CD3CAR NK cells. The broad CD3+ population is alsoaffected by the CD3CAR NK cells.

FIG. 37, CD3CAR NK cells are able to lyse normal T cells as expected.Normal T cells were isolated from umbilical cord blood and transducedwith lentiviruses expressing GFP. The transduced GFP T cells were usedto co-culture with CD3CAR NK cells. Co-culture conditions were carriedout in NK cell media with 2.5% serum. Co-cultures were incubated for 24hours and labeled for flow cytometry analysis. The ability of CD3CAR NKcells to lyse target T cells was evaluated by comparing the amount ofresidual CD3+ GFP T-cells after co-culture. Importantly, with anincreased incubation period, target CD3+ GFP T-cells were shown to belysed with over 80% efficiency at a dosage of 5:1 effector to targetcell ratio.

FIGS. 38A-38B. CD3CAR NK cells demonstrate profound anti-leukemiceffects in vivo. A, NSG mice were sublethally irradiated and, after 24hours, intravenously injected with 1×10⁶ luciferase-expressing Jurkatcells (Day 0) to induce measurable tumor formation. On day 3 and 4 micewere intravenously injected with 5×10⁶ CD3CAR NK cells or vector controlNK cells each day. These injections were repeated on Days 6 and 7, andagain on Day 10, for a total of 2.5×10⁷ cells per mouse. (FIG. 38A) Ondays 4, 7, 9, and 13, mice were injected subcutaneously with RediJectD-Luciferin and subjected to IVIS imaging. FIG. 38B, Average lightintensity measured for the CD3CAR NK injected mice was compared to thatof vector control NK cell injected mice. C, Percentage of tumor cellskilled in mice treated with CD3CAR NK cells relative to control.

FIG. 39. Steps for generation of CAR T or NK cell targeting T-celllymphomas or T-cell leukemias.

FIG. 40. Three pairs of sgRNA per gene are designed with CHOPCHOP totarget CD2, CD3, CD5 and CD7. Three pairs of sgRNA were designed withCHOPCHOP to target the gene of interest. Gene-specific sgRNAs were thencloned into the lentiviral vector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre)expressing a human Cas9 and puromycin resistance genes linked with anE2A self-cleaving linker. The U6-sgRNA cassette is in front of the Cas9element. The expression of sgRNA and Cas9puro is driven by the U6promoter and SFFV promoter, respectively.

FIGS. 41A-41D. Generation of stable CD5-deficient CCRF-CEM and MOLT-4 Tcells using CRISPR/Cas9 lentivirus system. FIG. 41A. Flow cytometryanalysis demonstrating the loss of CD5 expression in CCRF-CEM T-cellswith CRISPR/Cas9 KD using two different sgRNAs,Lenti-U6-sgCD5a-SFFV-Cas9puro (sgCD5A) and Lenti-U6-sgCD5b-SFFV-Cas9puro(sgCD5B) after puromycin selection. Wild type control is seen in theleft most scatter plot. Because the CRISPR/Cas9 KD technique with sgRNACD5A was more successful at CD5 protein downregulation, this population(denoted by the blue circle and arrow) was selected for sorting,purification and analysis in FIG. 41B. FIG. 41B. Flow cytometry analysisdata indicating the percentage of purely sorted stable CD5 negativeCCRF-CEM cells transduced using the scCD5A CRISPR/Cas9 technique. Wenote the >99% purity of CD45 positive, CD5 negative CCRF sgCD5A T-cells.FIG. 41C. Flow cytometry analysis demonstrating the loss of CD5expression in MOLT-4 T-cells with CRISPR/Cas9 KD using two differentsgRNA sequences (sequence CD5A and CD5B, middle and right columns) afterpuromycin treatment. Wild type control is seen the leftmost scatterplot. Because the CRISPR/Cas9 KD technique with primer CD5A was moresuccessful at CD5 protein downregulation, this population (denoted bythe blue circle and arrow) was selected for sorting, purification andanalysis in FIG. 41D. FIG. 41D. Flow cytometry analysis data indicatingthe percentage of purely sorted stable CD5 negative MOLT-4 cellstransduced using the scCD5A CRISPR/Cas9 technique. We note the >99%purity of CD45 positive, CD5 negative MOLT-4 sgCD5A T-cells.

FIGS. 42A-42D. Generation and cell sorting of stable CD7 loss inCCRF-CEM cells or NK-92 cells using CRISPR/Cas9 lentivirus system. Thepercentage of CD7 loss in CCRF-CEM (FIGS. 42A and 42B) or NK-92(FIGS.42C and 42D) using sgCD7A (Lenti-U6-sgCD7a-SFFV-Cas9-puro) and sgCD7B(Lenti-U6-sgCD7b-SFFV-Cas9-puro) was determined by flow cytometricanalysis with CD45 and CD7 antibodies after puromycin treatment. Thevalues of insert in figures showed percentage of positive and negativeexpressing CD45 or CD7 among analysis. Right panel indicated thepercentage purity of sorted stable CD7 negative cells in CCRF-CEM (FIG.42B) or in NK-92 cells (FIG. 42D) prepared from CD7 negative cellstransduced using sgCD7A or sgCD7D CRISPR lentiviruses.

FIGS. 43A-43B. CD7CAR NK⁷⁻-92 cells effectively lyse T cell ALL cellline T cells that express CD7.To avoid self-killing, CD7 deficient NK-92(NK⁷⁻-92) cells were generated and transduced with CD7CAR. Twotransduced preparations of CD7CAR NK⁷⁻-92 cells, #A and #B were used totest their killing ability. A, Flow cytometry analysis of CCRF-CEM cellsalone (left column), in co-culture with GFP NK⁷⁻-92 cells, and inco-culture with CD7CAR-NK-92-cells, #A and B#. FIG. 43B, bar graphsbased on data obtained from FIG. 43A.

FIG. 44. CD3 multimeric protein complex. CD3 includes a protein complexand is composed of four distinct chains as described the figure above.The complex includes a CD3ζ chain, a CD3γ chain, and two CD3ε chains.These chains associate with the T-cell receptor (TCR) composing of αβchains.

DETAILED DESCRIPTION

The disclosure provides chimeric antigen receptor (CAR) compositions,methods and making thereof, and methods of using the CAR compositions.

Compositions Chimeric Antigen Receptor Polypeptides

In one embodiment the disclosure provides a chimeric antigen receptor(CAR) polypeptide having a signal peptide, an antigen recognitiondomain, a hinge region, a transmembrane domain, at least oneco-stimulatory domain, and a signaling domain.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound having amino acid residuescovalently linked by peptide bonds. A protein or peptide must contain atleast two amino acids, and no limitation is placed on the maximum numberof amino acids that can include a protein's or peptide's sequence.Polypeptides include any peptide or protein having two or more aminoacids joined to each other by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides, and oligomers, for example, and tolonger chains, which generally are referred to in the art as proteins,of which there are many types. “Polypeptides” include, for example,biologically active fragments, substantially homologous polypeptides,oligopeptides, homodimers, heterodimers, variants of polypeptides,modified polypeptides, derivatives, analogs, fusion proteins, amongothers. The polypeptides include natural peptides, recombinant peptides,synthetic peptides, or a combination thereof.

A “signal peptide” includes a peptide sequence that directs thetransport and localization of the peptide and any attached polypeptidewithin a cell, e.g. to a certain cell organelle (such as the endoplasmicreticulum) and/or the cell surface.

The signal peptide is a peptide of any secreted or transmembrane proteinthat directs the transport of the polypeptide of the disclosure to thecell membrane and cell surface, and provides correct localization of thepolypeptide of the present disclosure. In particular, the signal peptideof the present disclosure directs the polypeptide of the presentdisclosure to the cellular membrane, wherein the extracellular portionof the polypeptide is displayed on the cell surface, the transmembraneportion spans the plasma membrane, and the active domain is in thecytoplasmic portion, or interior of the cell.

In one embodiment, the signal peptide is cleaved after passage throughthe endoplasmic reticulum (ER), i.e. is a cleavable signal peptide. Inan embodiment, the signal peptide is human protein of type I, II, III,or IV. In an embodiment, the signal peptide includes an immunoglobulinheavy chain signal peptide.

The “antigen recognition domain” includes a polypeptide that isselective for an antigen, receptor, peptide ligand, or protein ligand ofthe target; or a polypeptide of the target.

The target specific antigen recognition domain preferably includes anantigen binding domain derived from an antibody against an antigen ofthe target, or a peptide binding an antigen of the target, or a peptideor protein binding an antibody that binds an antigen of the target, or apeptide or protein ligand (including but not limited to a growth factor,a cytokine, or a hormone) binding a receptor on the target, or a domainderived from a receptor (including but not limited to a growth factorreceptor, a cytokine receptor or a hormone receptor) binding a peptideor protein ligand on the target. The target includes CD2, CD3, CD4, CD5,CD7, CD8, and CD52. In another embodiment, the target includes anyportion of CD2, CD3, CD4, CD5, CD7, CD8, and CD52. In one embodiment,the target includes surface exposed portions of the CD2, CD3, CD4, CD5,CD7, CD8, and CD52 polypeptides.

In another embodiment, the target is the extracellular domain of CD2(SEQ ID NO. 19). In another embodiment, the target is the CD3 epsilonchain extracellular domain (SEQ ID NO. 20). In another embodiment, thetarget is the CD4 extracellular domain (SEQ ID NO. 21). In anotherembodiment, the target is the CD5 extracellular domain (SEQ ID NO. 22).In another embodiment, the target is the CD7 extracellular domain (SEQID NO. 23). In another embodiment, the target is the CD8 alpha chainextracellular domain (SEQ ID NO. 24). In another embodiment, the targetis the CD8 beta chain extracellular domain (SEQ ID NO. 25). In anotherembodiment, the target is the CD52 CAMPATH-1 antigen (SEQ ID NO. 26).

In one embodiment, the antigen recognition domain includes the bindingportion or variable region of a monoclonal or polyclonal antibodydirected against (selective for) the target.

In one embodiment, the antigen recognition domain includes fragmentantigen-binding fragment (Fab). In another embodiment, the antigenrecognition domain includes a single-chain variable fragment (scFV).scFV is a fusion protein of the variable regions of the heavy (VH) andlight chains (VL) of immunoglobulins, connected with a short linkerpeptide.

In another embodiment, the antigen recognition domain includes Camelidsingle domain antibody, or portions thereof. In one embodiment, Camelidsingle-domain antibodies include heavy-chain antibodies found incamelids, or VHH antibody. A VHH antibody of camelid (for example camel,dromedary, llama, and alpaca) refers to a variable fragment of a camelidsingle-chain antibody (See Nguyen et al, 2001; Muyldermans, 2001), andalso includes an isolated VHH antibody of camelid, a recombinant VHHantibody of camelid, or a synthetic VHH antibody of camelid.

In another embodiment, the antigen recognition domain includes ligandsthat engage their cognate receptor. In another embodiment, the antigenrecognition domain is humanized.

It is understood that the antigen recognition domain may include somevariability within its sequence and still be selective for the targetsdisclosed herein. Therefore, it is contemplated that the polypeptide ofthe antigen recognition domain may be at least 95%, at least 90%, atleast 80%, or at least 70% identical to the antigen recognition domainpolypeptide disclosed herein and still be selective for the targetsdescribed herein and be within the scope of the disclosure.

In another embodiment, the antigen recognition domain is selective forSEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO.23, SEQ ID NO. 24, or SEQ ID NO. 25, or SEQ ID NO. 26.

The hinge region is a sequence positioned between for example,including, but not limited to, the chimeric antigen receptor, and atleast one co-stimulatory domain and a signaling domain. The hingesequence may be obtained including, for example, from any suitablesequence from any genus, including human or a part thereof. Such hingeregions are known in the art. In one embodiment, the hinge regionincludes the hinge region of a human protein including CD-8 alpha, CD28,4-1BB, OX40, CD3-zeta, T cell receptor α or β chain, a CD3 zeta chain,CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64,CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof,and combinations thereof.

In one embodiment the hinge region includes the CD8 a hinge region.

In some embodiments, the hinge region includes one selected from, but isnot limited to, immunoglobulin (e.g. IgG1, IgG2, IgG3, IgG4, and IgD).

The transmembrane domain includes a hydrophobic polypeptide that spansthe cellular membrane. In particular, the transmembrane domain spansfrom one side of a cell membrane (extracellular) through to the otherside of the cell membrane (intracellular or cytoplasmic).

The transmembrane domain may be in the form of an alpha helix or a betabarrel, or combinations thereof. The transmemebrane domain may include apolytopic protein, which has many transmembrane segments, eachalpha-helical, beta sheets, or combinations thereof.

In one embodiment, the transmembrane domain that naturally is associatedwith one of the domains in the CAR is used. In another embodiment, thetransmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

For example, a transmembrane domain includes a transmembrane domain of aT-cell receptor α or β chain, a CD3 zeta chain, CD28, CD3ε, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,ICOS, CD154, functional derivatives thereof, and combinations thereof.

The artificially designed transmembrane domain is a polypeptide mainlycomprising hydrophobic residues such as leucine and valine. In oneembodiment, a triplet of phenylalanine, tryptophan and valine is foundat each end of the synthetic transmembrane domain.

In one embodiment, the transmembrane domain is the CD8 transmembranedomain. In another embodiment, the transmembrane domain is the CD28transmembrane domain. Such transmembrane domains are known in the art.

The signaling domain and co-stimulatory domain include polypeptides thatprovide activation of an immune cell to stimulate or activate at leastsome aspect of the immune cell signaling pathway.

In an embodiment, the signaling domain includes the polypeptide of afunctional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fcgamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10),DNAX-activating protein 12 (DAP12), active fragments thereof, functionalderivatives thereof, and combinations thereof. Such signaling domainsare known in the art.

In an embodiment, the CAR polypeptide further includes one or moreco-stimulatory domains. In an embodiment, the co-stimulatory domain is afunctional signaling domain from a protein including OX40, CD27, CD28,CD30, CD40, PD-1, CD2, CD7, CD258, Natural killer Group 2 member C(NKG2C), Natural killer Group 2 member D (NKG2D), B7-H3, a ligand thatbinds to CD83, ICAM-1, LFA-1 (CD1 la/CD18), ICOS and 4-1BB (CD137),active fragments thereof, functional derivatives thereof, andcombinations thereof.

In one embodiment, the CAR polypeptide is CD2CAR, and includes SEQ IDNO. 10 or SEQ ID NO. 11. In one embodiment, the CAR polypeptide isCD3CAR, and includes SEQ ID NO. 12. In one embodiment, the CARpolypeptide is CD4CAR, and includes SEQ ID NO. 13 or SEQ ID NO. 14. Inone embodiment, the CAR polypeptide is CD5CAR, and includes SEQ ID NO.15. In one embodiment, the CAR polypeptide is CD7CAR, and includes SEQID NO. 17.In one embodiment, the CAR polypeptide is CD52CAR, andincludes SEQ ID NO. 18.

Polynucleotide Encoding Chimeric Antigen Receptor

The present disclosure further provides a polynucleotide encoding thechimeric antigen receptor polypeptide described above. Thepolynucleotide encoding the CAR is easily prepared from an amino acidsequence of the specified CAR by any conventional method. A basesequence encoding an amino acid sequence can be obtained from theaforementioned NCBI RefSeq IDs or accession numbers of GenBenk for anamino acid sequence of each domain, and the nucleic acid of the presentdisclosure can be prepared using a standard molecular biological and/orchemical procedure. For example, based on the base sequence, apolynucleotide can be synthesized, and the polynucleotide of the presentdisclosure can be prepared by combining DNA fragments which are obtainedfrom a cDNA library using a polymerase chain reaction (PCR).

In one embodiment, the polynucleotide disclosed herein is part of agene, or an expression or cloning cassette.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Polynucleotide includes DNA and RNA. Furthermore, nucleicacids are polymers of nucleotides. Thus, nucleic acids andpolynucleotides as used herein are interchangeable. One skilled in theart has the general knowledge that nucleic acids are polynucleotides,which can be hydrolyzed into the monomeric “nucleotides.” The monomericnucleotides can be hydrolyzed into nucleosides. As used hereinpolynucleotides include, but are not limited to, all nucleic acidsequences which are obtained by any means available in the art,including, without limitation, recombinant means, i.e., the cloning ofnucleic acid sequences from a recombinant library or a cell genome,using ordinary cloning technology and polymerase chain reaction (PCR),and the like, and by synthetic means.

In one embodiment, the polynucleotide includes the CD2CAR polynucleotideof SEQ ID NO. 1 or SEQ ID NO. 2. In one embodiment, the polynucleotideincludes the CD3CAR polynucleotide of SEQ ID NO. 3. In one embodiment,the polynucleotide includes the CD4CAR polynucleotide of SEQ ID NO. 4 orSEQ ID NO. 5. In one embodiment, the polynucleotide includes the CD5CARpolynucleotide of SEQ ID NO. 6. In one embodiment, the polynucleotideincludes the CD7CAR polynucleotide of SEQ ID NO. 8. In one embodiment,the polynucleotide includes the CD52CAR polynucleotide of SEQ ID NO. 9.

Polynucleotide Vector

The polynucleotide described above can be cloned into a vector. A“vector” is a composition of matter which includes an isolatedpolynucleotide and which can be used to deliver the isolatedpolynucleotide to the interior of a cell. Numerous vectors are known inthe art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, phagemid, cosmid, and viruses. Viruses include phages, phagederivatives. Thus, the term “vector” includes an autonomouslyreplicating plasmid or a virus. The term should also be construed toinclude non-plasmid and non-viral compounds which facilitate transfer ofnucleic acid into cells, such as, for example, polylysine compounds,liposomes, and the like. Examples of viral vectors include, but are notlimited to, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like.

In one embodiment, vectors include cloning vectors, expression vectors,replication vectors, probe generation vectors, integration vectors, andsequencing vectors.

In an embodiment, the vector is a viral vector. In an embodiment, theviral vector is a retroviral vector or a lentiviral vector. In anembodiment, the engineered cell is virally transduced to express thepolynucleotide sequence.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, adenovirus vectorsare used. A number of adenovirus vectors are known in the art. In oneembodiment, lentivirus vectors are used.

Viral vector technology is well known in the art and is described, forexample, in Sambrook et al, (2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York), and in other virologyand molecular biology manuals. Viruses, which are useful as vectorsinclude, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendomiclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Expression of chimeric antigen receptor polynucleotide may be achievedusing, for example, expression vectors including, but not limited to, atleast one of a SFFV or human elongation factor 11α (EF) promoter, CAG(chicken beta-actin promoter with CMV enhancer) promoter humanelongation factor 1α (EF) promoter. Examples ofless-strong/lower-expressing promoters utilized may include, but is notlimited to, the simian virus 40 (SV40) early promoter, cytomegalovirus(CMV) immediate-early promoter, Ubiquitin C (UBC) promoter, and thephosphoglycerate kinase 1 (PGK) promoter, or a part thereof. Inducibleexpression of chimeric antigen receptor may be achieved using, forexample, a tetracycline responsive promoter, including, but not limitedto, TRE3GV (Tet-response element, including all generations andpreferably, the 3rd generation), inducible promoter (ClontechLaboratories, Mountain View, Calif.) or a part or a combination thereof.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor—1 a(EF-1 a). However, other constitutive promoter sequences may also beused, including, but not limited to the simian virus 40 (SV40) earlypromoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avianleukemia virus promoter, an Epstein-Barr virus immediate early promoter,a Rous sarcoma virus promoter, as well as human gene promoters such as,but not limited to, the actin promoter, the myosin promoter, thehemoglobin promoter, and the creatine kinase promoter. Further, thedisclosure should not be limited to the use of constitutive promoters,inducible promoters are also contemplated as part of the disclosure. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metalothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorincludes sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide,

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-100 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another, in the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription,

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors, in other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle). In the casewhere a non-viral delivery system is utilized, an exemplary deliveryvehicle is a liposome. The use of lipid formulations is contemplated forthe introduction of the nucleic acids into a host cell (in vitro, exvivo or in vivo). In another aspect, the nucleic acid may be associatedwith a lipid. The nucleic acid associated with a lipid may beencapsulated in the aqueous interior of a liposome, interspersed withinthe lipid bilayer of a liposome, attached to a liposome via a linkingmolecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyi phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyi phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol.

“Liposome” is a generic term encompassing a variety of single andmultilamellar lipid vehicles formed by the generation of enclosed lipidbilayers or aggregates. Liposomes can be characterized as havingvesicular structures with a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh etal., 19 1 Glycobiology 5; 505-10). However, compositions that havedifferent structures in solution than the normal vesicular structure arealso encompassed. For example, the lipids may assume a micellarstructure or merely exist as nonuniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous polynucleotidesinto a host cell or otherwise expose a cell to the polynucleotide of thepresent disclosure, in order to confirm the presence of the recombinantDNA sequence in the host cell, a variety of assays may be performed.Such assays include, for example, “molecular biological” assays wellknown to those of skill in the art, such as Southern and Northernblotting, RT-PCR and PCR; “biochemical” assays, such as detecting thepresence or absence of a particular peptide, e.g., by immunologicalmeans (ELISAs and Western blots) or by assays described herein toidentify agents falling within the scope of the disclosure.

Engineered Cell

In another embodiment, the disclosure provides an engineered cellexpressing the chimeric antigen receptor polypeptide described above orpolynucleotide encoding for the same, and described above.

An “engineered cell” means any cell of any organism that is modified,transformed, or manipulated by addition or modification of a gene, a DNAor RNA sequence, or protein or polypeptide. Isolated cells, host cells,and genetically engineered cells of the present disclosure includeisolated immune cells, such as NK cells and T cells that contain the DNAor RNA sequences encoding a chimeric antigen receptor or chimericantigen receptor complex and express the chimeric receptor on the cellsurface. Isolated host cells and engineered cells may be used, forexample, for enhancing an NK cell activity or a T lymphocyte activity,treatment of cancer, and treatment of infectious diseases.

Any cell capable of expressing and/or capable of integrating thechimeric antigen receptor polypeptide, as disclosed herein, into itsmembrane may be used.

In an embodiment, the engineered cell includes immunoregulatory cells.Immunoregulatory cells include T-cells, such as CD4 T-cells (HelperT-cells), CD8 T-cells (Cytotoxic T-cells, CTLs), and memory T cells ormemory stem cell T cells. In another embodiment, T-cells include NaturalKiller T-cells (NK T-cells).

In an embodiment, the engineered cell includes Natural Killer cells.Natural killer cells are well known in the art. In one embodiment,natural killer cells include cell lines, such as NK-92 cells. Furtherexamples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, andNKL cells.

NK cells mediate anti-tumor effects without the risk of GvHD and areshort-lived relative to T-cells. Accordingly, NK cells would beexhausted shortly after destroying cancer cells, decreasing the need foran inducible suicide gene on CAR constructs that would ablate themodified cells.

In one embodiment, the engineered cell may include more than one typechimeric antigen receptor polypeptide described herein. Embodimentswherein the engineered cell includes at least two of a CD2CAR, CD3CAR,CD4CAR, CD5CAR, CD7CAR, CD8CAR, and CD52CAR have been contemplated. Forexample, the engineered cell may include a CD4 chimeric antigen receptorpolypeptide (CD4CAR) and a CD5 chimeric antigen receptor polypeptide(CD5CAR).

As used herein, CDXCAR refers to a chimeric antigen receptor having aCDX antigen recognition domain. As used herein CDX may be any one ofCD2, CD3, CD4, CD5, CD7, CD8, and CD52.

TCR Deficient T Cells Used to Carry CAR

In one embodiment, engineered cells, in particular allogeneic T cellsobtained from donors can be modified to inactivate components of TCR (Tcell receptor) involved in MHC recognition. As a result, TCR deficient Tcells would not cause graft versus host disease (GVHD).

T-Antigen Deficient T and NK Cells

T cell lymphomas or T cell leukemias express specific antigens, whichmay represent useful targets for these diseases. For instance, T celllymphomas or leukemias express CD7, CD2, CD3 and CD5. However, CD7, CD2,CD3, and CD5 are also expressed in CAR T or NK cells (except for CD3 andCD5), which offset their ability of targeting these antigens. Theself-killing might occur in T cells or NK cells armed with CARstargeting any one of these antigens. This makes generation of CARstargeting these antigens difficult. Therefore, it may be necessary toinactivate an endogenous antigen in a T or NK cell when it is used as atarget to arm CARs.

In another embodiment, the engineered cell is further modified toinactivate cell surface polypeptide to prevent engineered cells fromacting on other engineered cells. For example, one or more of theendogenous CD2, CD3, CD4, CD5, and CD7 genes of the engineered cells maybe knocked out or inactivated. In a preferred embodiment, the engineeredcell is a natural killer cell having at least one of the endogenous CD2and CD7 genes knocked out or inactivated.

In another preferred embodiment, the engineered cell is a T-cell havingat least one of the endogenous CD2, CD3, CD4, CD5, CD7, and CD8 genesknocked out or inactivated. In another preferred embodiment, theengineered cell is a NK cell having at least one of the endogenous CD2and CD7 genes knocked out or inactivated.

In one embodiment, the engineered cell expressing a CAR having aparticular antigen recognition domain will have the gene expressing thatantigen inactivated or knocked out. For example, a T-cell having a CD2CAR will have an inactivated or knocked out CD2 antigen gene. In anotherembodiment, an engineered cell (e.g. NK cell or T-cell) having a CARwith a CD4 antigen recognition domain will be modified so that the CD4antigen is not expressed on its cell surface. In another embodiment, anengineered cell (e.g. NK cell or T-cell) having one CAR with a CD2antigen recognition domain and another CAR with a CD7 antigenrecognition domain may have both the CD2 antigen gene and the CD7antigen gene knocked out or inactivated.

TABLE 1 cell surface antigens of Natural Killer cells and T-cells.Natural Killer cells T-cells CD2 + + CD4 − + CD3 − + CD5 − + CD7 + + CD8− +

Methods to knock out or inactivate genes are commonly known in the art.For example, CRISPR/Cas9 system, zinc finger nuclease (ZFNs) and TALEnucleases (TALENs) and meganucleases may be used to knock out orinactivate the CD2, CD3, CD4, CD5, CD7, CD8, and CD52 genes of theengineered cells.

Sources of Cells

The engineered cells may be obtained from peripheral blood, cord blood,bone marrow, tumor infiltrating lymphocytes, lymph node tissue, orthymus tissue. The host cells may include placental cells, embryonicstem cells, induced pluripotent stem cells, or hematopoietic stem cells.The cells may be obtained from humans, monkeys, chimpanzees, dogs, cats,mice, rats, and transgenic species thereof. The cells may be obtainedfrom established cell lines.

The above cells may be obtained by any known means. The cells may beautologous, syngeneic, allogeneic, or xenogeneic to the recipient of theengineered cells.

The term “autologous” refer to any material derived from the sameindividual to whom it is later to be re-introduced into the individual.

The term “allogeneic” refers to any material derived from a differentanimal of the same species as the individual to whom the material isintroduced. Two or more individuals are said to be allogeneic to oneanother when the genes at one or more loci are not identical. In someaspects, allogeneic material from individuals of the same species may besufficiently unlike genetically to interact antigenically.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “syngeneic” refers to an extremely close genetic similarity oridentity especially with respect to antigens or immunological reactions.Syngeneic systems include for example, models in which organs and cells(e.g. cancer cells and their non-cancerous counterparts) come from thesame individual, and/or models in which the organs and cells come fromdifferent individual animals that are of the same inbred strain.

Suicide System

The engineered cells of the present disclosure may also include asuicide system. Suicide systems provide a mechanism whereby theengineered cell, as described above, may be deactivated or destroyed.Such a feature allows precise therapeutic control of any treatmentswherein the engineered cells are used. As used herein, a suicide systemprovides a mechanism by which the cell having the suicide system can bedeactivated or destroyed. Suicide systems are well known in the art.

In one embodiment, a suicide system includes a gene that can bepharmacologically activated to eliminate the containing cells asrequired. In specific aspects, the suicide gene is not immunogenic tothe host harboring the polynucleotide or cell. In one example, thesuicide system includes a gene that causes CD20 to be expressed on thecell surface of the engineered cell. Accordingly, administration ofrituximab may be used to destroy the engineered cell containing thegene.

In some embodiments, the suicide system includes an epitope tag.Examples of epitope tags include a c-myc tag, streptavidin-bindingpeptide (SBP), and truncated EGFR gene (EGFRt). In this embodiment, theepitope tag is expressed in the engineered cell. Accordingly,administration of an antibody against the epitope tag may be used todestroy the engineered cell containing the gene.

In another embodiment, the suicide system includes a gene that causestruncated epidermal growth factor receptor to be expressed on thesurface of the engineered cell. Accordingly, administration of cetuximabmay be used to destroy the engineered cell containing the gene.

In another embodiment, the suicide gene may include caspace 8 gene,caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochromeP450.

Examples of further suicide systems include those described by Jones etal. (Jones B S, Lamb L S, Goldman F and Di Stasi A (2014) Improving thesafety of cell therapy products by suicide gene transfer. Front.Pharmacol. 5:254. doi: 10.3389/fphar.2014.00254), which is hereinincorporated by reference in its entirety.

CD2CAR

The CD2 adhesion molecule is a cell surface antigen expressed by allperipheral blood T cells and natural killer cells, but not on Blymphocytes. The extracellular domain of CD2 containsimmunoglobulin-like domains which can mediate homodimerization. Ligationof CD2 by CD58 (LFA-3) or CD48 helps T cells adhere toantigen-presenting cells, and triggers signal transduction pathways thatenhance signaling through the T cell receptor for antigen. CD2 knockoutmice exhibit normal immune function, and it is thought that CD2 issimilar functionally with other T cell co-stimulatory receptors such asCD28.

CD2 is expressed in T-ALL, T cell lymphoma/leukemia, acute promyelocyticleukemia (microgranular variant), systemic mastocytosis, mast celldisease, thymoma and acute myeloid lymphoma (M0) and NK cell leukemia.

In one embodiment, the disclosure provides a chimeric antigen receptorpolypeptide having an antigen recognition domain specific for a CD2antigen, and engineered cells expressing the same.

In another embodiment, the disclosure provides a chimeric antigenreceptor polypeptide having a variant of the sequence of an antigenrecognition domain specific for a CD2 antigen, and engineered cellsexpressing the same.

In one embodiment, the CD2 CAR includes at least one co-stimulatorydomain. In another embodiment, the CD2CAR includes at least twoco-stimulatory domains.

In one embodiment, the CD2CAR includes SEQ ID NO. 10 and SEQ ID NO. 11.

CD3CAR

CD3 consists of a protein complex and is composed of four distinctchains as described the figure above. The complex contains, a CD3ζchain, a CD3γ chain, and two CD3ε chains. These chains associate withthe T-cell receptor (TCR) composing of αβ chains.

The TCR/CD3 complex is a unique marker for T lineage cells. There is avariety of monoclonal antibodies against this complex that have beendeveloped. One such monoclonal antibody is the murine monoclonalantibody OKT3 against the surface CD3. CD3 is the common marker for Tcells and T cell malignancies. OKT3 against CD3 epsilon is the commonantibody used for identifying T cells. Anti-CD3 monoclonal antibodies astreatments include: (1) acute renal, cardiac or hepatic allograftrejection; (2) depletion of T cells from donor marrow prior totransplant; (3) new onset of type I diabetes. CD3 against CD3 epsilonchain is the most specific T cell antibody used to identify T cells inbenign and malignant disorders. CD3 is found in 86% of peripheral T celllymphomas.

In some embodiments, the disclosure includes a method for generation ofCD3CAR. In further embodiments, CD3CAR includes a scFv antibody whichspecifically binds to the surface protein of CD3.

In some embodiments, CD3CAR includes an scFv molecule, whichspecifically binds to the TCR/CD3 complexes.

In some embodiments, the scFv in the CAR may be a molecule specificallybinding to the extracellular domains of αβTCR associated with CD3.

CD4CAR

In one embodiment, chimeric antigen receptor of the present disclosureincludes a CD4 antigen recognition domain, CD4CAR.

In one embodiment, the CD4 CAR includes at least one co-stimulatorydomain. In another embodiment, the CD4CAR includes at least twoco-stimulatory domains.

In one embodiment, CD4CAR includes SEQ ID NO. 13 and SEQ ID NO. 14.

CD5CAR

In another embodiment, the disclosure provides a chimeric antigenreceptor polypeptide having an antigen recognition domain specific forCD5, and engineered cells expressing the same.

In one embodiment, the CD5CAR includes at least one-costimulatorydomain. In another embodiment, the CD5CAR includes at least twoco-stimulatory domains.

CD7CAR

CD7 is a transmembrane protein which is a member of the immunoglobulinsuperfamily. This protein is expressed on the surface of mature T cells.It is the earliest surface antigen expressed on T cell lineage cells.

CD7 is a very good marker for T-ALL and more than 90% of T-ALL expressCD7. CD7 is also expressed in NK lymphoma, T cell lymphoma/leukemia,chronic myeloid leukemia, acute myeloid leukemia, and lymphocyte richthymoma

In one embodiment, the disclosure provides a chimeric antigen receptorpolypeptide having an antigen recognition domain specific for a CD7antigen, and engineered cells expressing the same.

In one embodiment, the CD7CAR includes at least one co-stimulatorydomain. In another embodiment, the CD7CAR includes at least twoco-stimulatory domains

Methods Method of Making Engineered Cells

In one embodiment, the disclosure also provides methods of making theengineered cells described above.

In this embodiment, the cells described above are obtained or isolated.The cells may be isolated by any known means. The cells includeperipheral blood cells or cord blood cells. In another embodiment, thecells are placental cells, embryonic stem cells, induced pluripotentstem cells, or hematopoietic stem cells.

The polynucleotide encoding for the chimeric antigen receptorpolypeptide described above is introduced into the peripheral bloodcells or cord blood cells by any known means. In one example, thepolynucleotide encoding for the chimeric antigen receptor polypeptidedescribed above is introduced into the cell by way of viral vector.

The polynucleotide encoding for the chimeric antigen receptorpolypeptide described above is introduced into the placental cells,embryonic stem cells, induced pluripotent stem cells, or hematopoieticstem cells by any known means. In one example, the polynucleotideencoding for the chimeric antigen receptor polypeptide described aboveis introduced into the cell by way of viral vector.

In other embodiments, the chimeric antigen receptor polynucleotide maybe constructed as a transient RNA-modified “biodegradable derivatives”.The RNA-modified derivatives may be electroporated into a T cell or NKcell. In a further embodiment, chimeric antigen receptor describedherein may be constructed in a transponson system also called a“Sleeping Beauty”, which integrates the chimeric antigen receptorpolynucleotide into the host genome without a viral vector.

Once the polynucleotide described above is introduced into the cell toprovide an engineered cell, the engineered cells are expanded. Theengineered cells containing the polynucleotide described above areexpanded by any known means.

The expanded cells are isolated by any known means to provide isolatedengineered cells according to the present disclosure.

Methods of Using

The disclosure provides methods to kill, reduce the number of, ordeplete immunoregulatory cells. In another embodiment, the disclosureprovides a method to kill, reduce the number of, or deplete cells havingat least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

As used herein, “reduce the number of” includes a reduction by at least5%, at least 10%, at least 25%, at least 50%, at least 75%, at least80%, at least 90%, at least 99%, or 100%.

As used herein, “deplete” includes a reduction by at least 75%, at least80%, at least 90%, at least 99%, or 100%.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD2 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD2 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD2 may be determined by any cell deathassay known in the art.

As used herein, the immunoregulatory cells may be in a patient, in cellculture, or isolated.

As used herein, “patient” includes mammals. The mammal referred toherein can be any mammal. As used herein, the term “mammal” refers toany mammal, including, but not limited to, mammals of the orderRodentia, such as mice and hamsters, and mammals of the orderLogomorpha, such as rabbits. The mammals may be from the orderCarnivora, including Felines (cats) and Canines (dogs). The mammals maybe from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). Themammals may be of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). Preferably, the mammal is ahuman.

In certain embodiments, the patient is a human 0 to 6 months old, 6 to12 months old, 1 to 5 years old, 5 to 10 years old, 5 to 12 years old,10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old,35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old,70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90years old, 90 to 95 years old or 95 to 100 years old.

The terms “effective amount” and “therapeutically effective amount” ofan engineered cell as used herein mean a sufficient amount of theengineered cell to provide the desired therapeutic or physiological oreffect or outcome. Such, an effect or outcome includes reduction oramelioration of the symptoms of cellular disease. Undesirable effects,e.g. side effects, are sometimes manifested along with the desiredtherapeutic effect; hence, a practitioner balances the potentialbenefits against the potential risks in determining what an appropriate“effective amount” is. The exact amount required will vary from subjectto subject, depending on the species, age and general condition of thesubject, mode of administration and the like. Thus, it may not bepossible to specify an exact “effective amount”. However, an appropriate“effective amount” in any individual case may be determined by one ofordinary skill in the art using only routine experimentation. Generally,the engineered cell or engineered cells is/are given in an amount andunder conditions sufficient to reduce proliferation of target cells.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD2 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD2 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD2 may be determined by any cell deathassay known in the art.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD3 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD3 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD3 may be determined by any cell deathassay known in the art.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD4 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD4 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD4 may be determined by any cell deathassay known in the art.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD5 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD5 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD5 may be determined by any cell deathassay known in the art.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD7 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD7 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD7 may be determined by any cell deathassay known in the art.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having a CD8 antigen by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD8 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD8 may be determined by any cell deathassay known in the art.

In one embodiment, the disclosure includes a method of reducing thenumber of immunoregulatory cells having CD52 by contacting theimmunoregulatory cells with an effective amount of the engineered cellsdescribed above expressing a chimeric antigen receptor peptide having aCD52 antigen recognition domain. Optionally, the reduction in the numberof immunoregulatory cells having CD52 may be determined by any celldeath assay known in the art.

Method of Treatment

In another embodiment, the disclosure provides methods for the treatmentof a cell proliferative disease. The method includes administration of atherapeutically effective amount of the engineered cells described aboveto a patient in need thereof.

Cell proliferative disease is any one of cancer, neoplastic disease orany disease involving uncontrolled cell proliferation (e.g. formation ofcell mass) without any differentiation of those cells into specializedand different cells.

Cell proliferative diseases as also include a malignancy, or aprecancerous condition such as a myelodysplasia syndrome or apreleukemia, or prelymphoma.

With respect to the disclosed methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma),bone cancer, brain cancer (e.g., meduUoblastoma), breast cancer, cancerof the anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, headand neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkinlymphoma, hypopharynx cancer, kidney cancer, larynx cancer, leukemia,liquid tumors, liver cancer, lung cancer (e.g., non-small cell lungcarcinoma), lymphoma, malignant mesothelioma, mastocytoma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, B-chroniclymphocytic leukemia, hairy cell leukemia, acute lymphoblastic leukemia(ALL), T-cell acute lymphocytic leukemia, and Burkitt's lymphoma,extranodal NK/T cell lymphoma, NK cell leukemia/lymphoma,post-transplant lymphoproliferative disorders, ovarian cancer,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, solid tumors, stomach cancer,testicular cancer, thyroid cancer, and ureter cancer. Preferably, thecancer is a hematological malignancy (e.g., leukemia or lymphoma,including but not limited to Hodgkin lymphoma, non-Hodgkin lymphoma,chronic lymphocytic leukemia, acute lymphocytic cancer, acute myeloidleukemia, B-chronic lymphocytic leukemia, hairy cell leukemia, acutelymphoblastic leukemia (ALL), and Burkitt's lymphoma), thymic carcinoma,diffuse large cell lymphoma, mantle cell lymphoma, small lymphocyticlymphoma (SLL), and chronic lymphoid leukemia(CLL), T-cell lymphoma, andperipheral T-cell lymphoma.

The disclosure provides a method for the treatment of acute organrejection by depletion of T and NK cells that are associated with CD2,CD3, CD4, CD5, CD7, CD8, and CD52.

In one embodiment, the disclosure incudes a method for the treatment ofacute or chronic graft versus host disease (GVHD) by depletion of Tcells and NK cells that are associated with at least one of CD2, CD3,CD4, CD5, CD7, CD8, and CD52.

In one embodiment, the disclosure provides a method to prevent organrejection by administering to a patient who has undergone organtransplant or will undergo an organ transplant an effective amount of anengineered cell having CD3CAR.

In another embodiment, the disclosure provides a method to prevent ortreat GVHD by administering to a patient in need thereof an effectiveamount of an engineered cell having CD3CAR.

In one embodiment, the disclosure incudes a method for the depletion orreduction of donor and host T or NK cells using CAR T or NK cells invivo for stem cell transplant. This could be accomplished byadministration of CAR T or NK cells to a patient immediately before theinfusion of the bone marrow stem cell graft.

The disclosure provides a method of immunotherapy as a conditioning orbridge-to-transplant strategy or stand-alone for the treatment of cellproliferative diseases that are associated with at least one of CD2,CD3, CD4, CD5, CD7, CD8, and CD52.

The disclosure provides a method for the treatment of cell proliferativediseases that are associated with at least one of CD2, CD3, CD4, CD5,CD7, CD8, and CD52.

In another embodiment, the disclosure provides a method for thetreatment of non-cancer related diseases that are associated with theexpression of at least one of CD2, CD3, CD4, CD5, CD7, CD8, and CD52.

In some embodiments, CAR having a CD2, CD3, CD4, CD5, CD7, CD8, or CD52antigen recognition domain for use in the treatment of a cellproliferative disease is combined with a checkpoint blockade, such asCTLA-4 and PD1/PD-L1. This may lead to enhanced tumor eradication.

The presence of the immunosuppressive microenvironments can limit thefull functions of CAR T/NK cells. In some embodiments, the combinationof CD4CAR with checkpoint blockade such as CTLA-4 and PD1/PD-L1 can leadto enhanced tumor eradication. Currently checkpoint blockade is beingtested in clinical trials in combination with CAR T cells.

In some embodiments, CARs having a CD2, CD3, CD4, CD5, CD7, CD8, or CD52antigen recognition domain are used as a strategy to deepen, remove,reduce, resist and/or prolong responses to initial chemotherapy, or whencombined with other adjunct therapies. All available adjunct therapiesto treat or prevent the disease condition are considered to be part ofthis disclosure and are within the scope of the present disclosure

In some embodiments, NK cell CARs having a CD2, CD3, CD4, CD5, CD7, CD8,or CD52 antigen recognition domain, are administrated “off-the-shelf” toany mammal with cancer and/or autoimmune disorders.

CD3CAR

In some embodiments, the NK cell bearing the CD3 CAR exhibits anantitumor immunity and exerts the efficacy of killingleukemias/lymphomas expressing CD3

The disclosure provides methods for deleting or reducing abnormal ormalignant T cells in bone marrow, blood and organs using CD3CAR NKcells. In some embodiments, CD3 positive malignancies may include, butis not limited to precursor T lymphoblastic leukemia/lymphoma, mature Tcell lymphomas/leukemias, EBV-positive T-cell lymphoproliferativedisorders, adult T-cell leukemia/lymphoma, mycosis fungoides/sezarysyndrome, primary cutaneous CD30-positive T-cell lymphoproliferativedisorders, peripheral T-cell lymphoma (not otherwise specified),angioimmunoblastic T-cell lymphoma and anaplastic large cell lymphoma.

In some embodiments, CD3CAR NK cells can be used to treat patients withT-leukemias/lymphomas, who are not eligible for stem cell therapy ornever achieved a remission despite many intensive chemotherapy regimens.In further embodiments, CD3CAR NK cells may be used as a component ofconditioning regimen for a bone marrow transplant or a bridge to thebone marrow transplant.

CD4CAR

In one embodiment, the engineered cell having the CD4CAR exhibits anantitumor immunity when the antigen recognition domain of the CAR bindsto its corresponding antigen. In a preferred embodiment, the CD8 T cellcomprising the CAR exerts the efficacy of killing leukemias/lymphomascells expressing CD4.

The present disclosure includes methods for deleting, reducing,treating, preventing or eliminating abnormal or malignant T cells foundin, including, but not limited to, bone marrow, blood, and/or organs. Insome embodiments, malignant CD4 expressing cells are present in patientswith precursor T lymphoblastic leukemia/lymphoma, mature T-celllymphomas/leukemias cells such as, for example, T-cell prolymphocyticleukemia, EBV-positive T cell lymphoproliferative disorders, adultT-cell leukemia/lymphoma, mycosis fungoides/sezary syndrome, primarycutaneous CD30-positive T-cell lymphoproliferative disorders, peripheralT-cell lymphoma (not otherwise specified), angioimmunoblastic T-celllymphoma, and anaplastic large cell lymphoma.

In some embodiments, CD4CAR cells are used to treatT-leukemias/lymphomas cells in patients not eligible for stem celltherapy or patients that have never achieved a remission despite manychemotherapy regimens.

In some embodiments, CD4CAR cells are used to treat CD4 expressing acutemyelomonocytic leukemia, acute monoblastic leukemia, monocytic leukemia,and chronic myelomonocytic leukemia.

In some embodiments, the CD4CAR T cells can be expanded in the T cellculture medium and the subpopulations such central memory T cells ornaïve T cells can be isolated and used to improved engraftment. Thesecells may persist and support memory T cell functions, which would makethem ideal candidates for long-term control of cancers

The presence of the immunosuppressive microenvironments can limit thefull functions of CAR T/NK cells. In some embodiments, the combinationof CD4CAR with checkpoint blockade such as CTLA-4 and PD1/PD-L1 can leadto enhanced tumor eradication.

In some embodiments, CD4CAR cells are used as a strategy to deepen,remove, reduce, resist and/or prolong responses to initial chemotherapy,or when combined with other adjunct therapies. All available adjuncttherapies to treat or prevent the disease condition are considered to bepart of this disclosure and are within the scope of the presentdisclosure. Chemotherapy includes, but is not limited to, CHOP(cyclophosphamide, doxorubicin, vincristie, prednisone), EPOCH(etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), orany other multidrug regimens. In a preferred embodiment, CD4CAR cellsare utilized for treating or preventing a residual disease after stemcell transplant and/or chemotherapy.

In one embodiment, the cell including the CD4CAR exhibits depletion ofimmunoregulatory cells when the antigen recognition domain of the CARbinds to its corresponding antigen. For example, the cells includingCD4CAR include, but are not limited to, at least one of CD8 T cell, NKcell, or NK-92 cell. Any other suitable cell having CD4CAR that exhibitand/or exerts the high efficacy of deletion of CD4 helper cells whenencountering them, whereby organ transplant rejections can be preventedor autoimmune diseases can be controlled or relieved is considered to bepart of this disclosure and within the scope of the present disclosure.

There is no concern about persisting CAR-associated side effectsobserved in CAR T cells. In some embodiments, CD4CAR NK cells may beadministrated to patients with autoimmune disorders in an acute orcritical clinical setting to rapidly deplete immunoregulatory cells suchas CD4 helper T cells, and thereby enable or allow new or non-memory CD4helper T cells to regenerate.

The disclosure includes a method of generating CD4CAR. In someembodiments, CD4CAR is generated using T-cells. In other embodiments,CD4CAR is generated using NK cells or NK-92 cells, such that they areadministered “off-the-shelf” to any mammal with cancer and/or autoimmunedisorders. In some embodiments, CD4CAR NK-92 or NK cells are able tokill cells, reduce, deplete, and/or prevent particular CD4+ T cells orcancer cells expressing CD4.

In some embodiments, CD4CAR NK-92 cells can be generated having a highlevel of expression of CD4CAR by flow cytometry using goat-anti-mouseFab antibodies or a part thereof. Any other type of antibody generatedusing any other genus is considered to be part of this disclosure and iswithin the scope of the present disclosure.

In some embodiments, CD4CAR NK-92 cells can be utilized for one therapyat a time when there is minimal residual disease after a stem celltransplant or chemotherapy.

In some embodiments, the CD4CAR is part of an expressing gene or acassette. In a preferred embodiment, the expressing gene or the cassettemay include an accessory gene or a epitope tag or a part thereof, inaddition to the CD4CAR. The accessory gene may be an inducible suicidegene or a part thereof, including, but not limited to, caspase 9 gene,thymidine kinase, cytosine deaminase (CD) or cytochrome P45029. The“suicide gene” ablation approaches improves safety of the gene therapyand kill cells only when activated by a specific compound or a molecule.In some embodiments, the suicide gene is inducible and is activatedusing a specific chemical inducer of dimerization (CID).

In some embodiments, the accessory tag is a c-myc tag, truncated EGFRgene (EGFRt) or a part or a combination thereof. The accessory tag maybe used as a nonimmunogenic selection tool or for tracking markers.

In some embodiments, the host cells expressing CD4CAR can beadministrated with one or more additional therapeutic agents to a mammal(e.g., a human). In this regard, the composition including the hostcells or the vector comprising CD4CAR can be administered first, and theone or more additional therapeutic agents can be administered second, orvice versa.

The present disclosure includes within its scope administering a typicalamount of host cells expressing CD4CAR to a mammal, which for examplemay be in the range from about 0.5 million to about 1 billion cells. Allsub-ranges and ranges outside the above-indicated range are consideredto be part of the disclosure and is within the scope of the presentdisclosure.

In a preferred embodiment, a SFFV promoter is used to redirect CD8 Tcells to target cells expressing CD4 and to drive CD4CAR expression. Insome embodiments, the CAR includes functional characteristics such as,extracellular expression of scFv and exertion of a strong immuneresponse when encountering with the CD4 expressing cells.

In one embodiment, the cell comprising the CD4CAR is selected from agroup including a cytotoxic T lymphocyte (CTL), and a Natural Killer(NK) cell. In a preferred embodiment, the cells having the CAR include,but are not limited to, CD8 T cells, NK cells, and NK-92 cells.

In some embodiments, CD4CAR may be used with drug conjugates, includingDNA/nucleic acid conjugates, peptides, chemical entities and/or smallmolecules to provide enhanced efficacy and safety.

Control of HIV-1 infection can be achieved in HIV patients using acombination of antiretroviral therapies, however, the viral loadincreases after discontinuation. The source or reservoir of re-emergentHIV-1 is memory CD4 T cells. In one embodiment, the CD4CAR of thepresent disclosure is used to deplete memory CD4 T cells, whereby asterilizing cure is accomplished for the HIV infection. In anotherembodiment, the CD4CAR assists in blocking HIV viral entrance, whereasCD4CAR binds to the CD4 protein, a protein essential for HIV entry.

Accordingly, the disclosure provides a method prevent organ transplantrejections by depleting CD4 T cells. The method includes administeringto a patient in need thereof a therapeutically effective amount of anengineered cell having a chimeric antigen receptor polypeptide having aCD4 antigen recognition domain.

CD5CAR

In another embodiment, administration of a CAR polypeptide having a CD5antigen recognition domain (CD5CAR) is used to treat rheumatoidarthritis. In another embodiment, CD5CAR may be used as a prophylaxisfor graft-versus-host disease following bone marrow transplantationtherapy (BMT) therapy. In another embodiment, CD5CAR may be used tomodify of CD5 expression in treatment of autoimmune disorders andmalignancies.

In some embodiments, the disclosure of engineered cell having a chimericantigen receptor selective for CD5 may act as a bridge to bone marrowtransplant for those patients who are not longer responding tochemotherapy or have minimal residual diseases and are not eligible forbone marrow transplant. In further embodiments, CD5CAR can eliminate CD5positive leukemic cells followed by bone marrow stem rescues to supportlymphopenia.

In particular embodiments, CD5CAR a T or NK cell targets cells thatexpress CD5. Target cells may be, but is not limited to cancer cells,such as T-cell lymphoma or T-cell leukemia, precursor acute T-celllymphoblastic leukemia/lymphoma, B cell chronic lymphocyticleukemia/small lymphocytic lymphoma, mantle cell lymphoma, CD5 positivediffuse large B cell lymphoma, and thymic carcinoma.

In one embodiment, CD5CAR may be used for treating non-hematologicdisorders including, but not limited to, rheumatoid arthritis,graft-versus-host-disease and autoimmune diseases.

The engineered or modified T cells may be expanded in the presence ofIL-2 or/and both IL-7 and IL-15, or using other molecules.

The introduction of CARs can be fulfilled before or after theinactivation of CD5 by expanding in vitro engineered T cells prior toadministration to a patient.

In particular embodiments, the inactivation of CD5 can be achieved byone of the following means:

(1) Expressing anti-CD5 scFv on T cell surface linked to a transmembranedomain via a hinge region. This may result in the conversion ofCD5-postive T cells to CD5 negative T cells.

(2). Expressing anti-CD5 scFv that specifically binds to CD5 protein ornegative modulators of CD5 thereof, or fragments or domains thereof.

In some embodiments, a scFv (single-chain antibody) against CD5 isderived from a monoclonal or polyclonal antibody binding tointracellular CD5 and blocks the transport of CD5 protein to the cellsurface. In a preferred embodiment, anti-CD5 scFv includes an ER(endoplasmic reticulum) retention sequence, KDEL. When it is expressedintracellularly and retained to the ER or Golgi, the anti-CD5 scFventraps CD5 within the secretion pathway, which results in theprevention of CD5 proper cell surface location in a T cell.

In some embodiments, CD5CAR T cells are co-administrated withimmunomodulatory drugs, such as, but not limited to CTLA-4 andPD-1/PD-L1 blockades, or cytokines, such as IL-2 and IL12 or inhibitorsof colony stimulating factor-1 receptor (CSF1R), such as FPA008, whichlead to better therapeutic outcomes.

In another embodiment, the disclosure provides a method of imparting,aiding, increasing, or boosting anti-leukemia or anti-lymphoma immunity.

The therapeutic agent including the engineered cell expressing the CARas an active ingredient can be administered intradermally,intramuscularly, subcutaneously, intraperitoneally, intranasally,intraarterially, intravenously, intratumorally, or into an afferentlymph vessel, by parenteral administration, for example, by injection orinfusion, although the administration route is not limited.

Any method of the disclosure may further includes the step of deliveringto the individual an additional cancer therapy, such as surgery,radiation, hormone therapy, chemotherapy, immunotherapy, or acombination thereof.

Chemotherapy includes, but is not limited to, CHOP (cyclophosphamide,doxorubicin, vincristie, prednisone), EPOCH (etoposide, vincristine,doxorubicin, cyclophosphamide, prednisone), or any other multidrugregimens. In a preferred embodiment, CD54CAR cells are utilized fortreating or preventing a residual disease after stem cell transplantand/or chemotherapy.

In another embodiment, any method of the disclosure may further includeantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further aspects, the T cells of the disclosure may be used in atreatment regimen in combination with chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies or otherantibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation. Drugsthat inhibit either the calcium dependent phosphatase calcineurin(cyclosporine and FK506) or inhibit the p70S6 kinase that is importantfor growth factor induced signaling (rapamycin). (Liu et al., Cell66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer etal., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a furtheraspect, the cell compositions of the present disclosure are administeredto a patient in conjunction with (e.g., before, simultaneously orfollowing) bone marrow transplantation, T cell ablative therapy usingeither chemotherapy agents such as, fludarabine, external-beam radiationtherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH.In one aspect, the cell compositions of the present disclosure areadministered following B-cell ablative therapy such as agents that reactwith CD20, e.g., Rituxan. For example, in one embodiment, subjects mayundergo standard treatment with high dose chemotherapy followed byperipheral blood stem cell transplantation. In certain embodiments,following the transplant, subjects receive an infusion of the expandedimmune cells of the present disclosure. In an additional embodiment,expanded cells are administered before or following surgery.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia greata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type 1),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, and ulcerative colitis.

The present disclosure may be better understood with reference to theexamples, set forth below. The following examples are put forth so as toprovide those of ordinary skill in the art with a complete disclosureand description of how the compounds, compositions, articles, devicesand/or methods claimed herein are made and evaluated, and are intendedto be purely exemplary and are not intended to limit the disclosure.

Following administration of the delivery system for treating,inhibiting, or preventing a cancer, the efficacy of the therapeuticengineered cell can be assessed in various ways well known to theskilled practitioner. For instance, one of ordinary skill in the artwill understand that a therapeutic engineered cell delivered inconjunction with the chemo-adjuvant is efficacious in treating orinhibiting a cancer in a subject by observing that the therapeuticengineered cell reduces the cancer cell load or prevents a furtherincrease in cancer cell load. Cancer cell loads can be measured bymethods that are known in the art, for example, using polymerase chainreaction assays to detect the presence of certain cancer cell nucleicacids or identification of certain cancer cell markers in the bloodusing, for example, an antibody assay to detect the presence of themarkers in a sample (e.g., but not limited to, blood) from a subject orpatient, or by measuring the level of circulating cancer cell antibodylevels in the patient.

Throughout this specification, quantities are defined by ranges, and bylower and upper boundaries of ranges. Each lower boundary can becombined with each upper boundary to define a range. The lower and upperboundaries should each be taken as a separate element.

Reference throughout this specification to “one embodiment,” “anembodiment,” “one example,” or “an example” means that a particularfeature, structure or characteristic described in connection with theembodiment or example is included in at least one embodiment of thepresent embodiments. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” “one example,” or “an example” invarious places throughout this specification are not necessarily allreferring to the same embodiment or example. Furthermore, the particularfeatures, structures or characteristics may be combined in any suitablecombinations and/or sub-combinations in one or more embodiments orexamples. In addition, it is appreciated that the figures providedherewith are for explanation purposes to persons ordinarily skilled inthe art and that the drawings are not necessarily drawn to scale.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but may include otherelements not expressly listed or inherent to such process, article, orapparatus.

Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “or”. For example, a condition Aor B is satisfied by any one of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

Additionally, any examples or illustrations given herein are not to beregarded in any way as restrictions on, limits to, or expressdefinitions of any term or terms with which they are utilized. Instead,these examples or illustrations are to be regarded as being describedwith respect to one particular embodiment and as being illustrativeonly. Those of ordinary skill in the art will appreciate that any termor terms with which these examples or illustrations are utilized willencompass other embodiments which may or may not be given therewith orelsewhere in the specification and all such embodiments are intended tobe included within the scope of that term or terms. Language designatingsuch nonlimiting examples and illustrations includes, but is not limitedto: “for example,” “for instance,” “e.g.,” and “in one embodiment.”

In this specification, groups of various parameters containing multiplemembers are described. Within a group of parameters, each member may becombined with any one or more of the other members to make additionalsub-groups. For example, if the members of a group are a, b, c, d, ande, additional sub-groups specifically contemplated include any one, two,three, or four of the members, e.g., a and c; a, d, and e; b, c, d, ande; etc.

Examples Targeting of Human T Cell Malignancies Using CD4-SpecificChimeric Antigen Receptor (CAR)-Engineered T Cells Materials and MethodsBlood Donors, Primary Tumor Cells and Cell Lines

Human lymphoma cells and peripheral blood mononuclear cells wereobtained from residual samples. Umbilical cord blood cells were obtainedfrom donors at Stony Brook University Hospital. SP53 and KARPAS 299lymphoma cell lines were obtained from ATCC (Manassas, Va.).

Lentivirus Production and Transduction of T Cells

To produce viral supernatant, 293FT cells were co-transfected with pMD2Gand pSPAX viral packaging plasmids, and with either pRSC.CD4.3G or GFPLentiviral vector, using Lipofectamine 2000 (Life Technologies,Carlsbad, Calif.) per manufacturer's protocol. Prior to lentiviraltransduction, umbilical cord or peripheral blood mononuclear buffy coatcells were activated for two days in the presence of 300 IU/mL IL-2 and1μg/mL anti-human CD3 (Miltenyi Biotec, Germany).

T Cell Expansion

CAR-transduced T cells were expanded for 7 days in T cell media (50%AIM-V, 40% RPMI 1640, 10% FBS and 1× penicillin/streptomycin; all Gibco)supplemented with IL-2. Cells were counted every day and media was addedevery 2-3 days in order to maintain T cell counts below 2×10⁶ cells/mL.

CAR Immunophenotype

For the analysis of CAR cell immunophenotype, following 7 days ofexpansion, CD4CAR T cells and GFP control cells were stained withCD45RO, CD45RA, CD62L and CD8 (all from BD Biosciences) for flowcytometry analysis.

Co-Culture Target Cell Ablation Assays

CD4CAR T cells or GFP T cells (control) were incubated with target cellsat ratios of 2:1, 5:1 and 10:1 (200,000, 500,000 or 1 million effectorcells to 100,000 target cells, respectively) in 1 mL T cell culturemedia, without IL-2 for 24 h. Target cells were KARPAS 299 cells(anaplastic large T cell lymphoma expressing CD4), leukemia cells from apatient with CD4+ T cell leukemia—Sezary syndrome—and from a patientwith CD4+ PTCL lymphoma. As a negative control, CD4CAR T cells and GFP Tcells were also incubated with SP53 (mantle cell lymphoma) cells, whichdo not express CD4, in the same ratios in 1 mL separate reactions. After24 hours of co-culture, cells were stained with mouse anti-human CD8 andCD4 antibodies. In the experiments with SP53 cells, SP53 cells werelabeled with CMTMR (Life Technologies) prior to co-culture with T cells,and T cells were labeled with mouse anti-human CD3 (PerCp) afterco-culture incubation.

In Vivo Mouse Xenogenic Model

NSG mice (NOD.Cg-Prkdc^(scid) Il2rg^(tmlWjl)/SzJ) from the JacksonLaboratory were used under a Stony Brook University IACUC-approvedprotocol. Mice were all male and between 8 and 12 weeks old. Three setsof in vivo experiments were performed with no blinding. For each set, 10mice were irradiated with a sublethal (2.5 Gy) dose of gamma irradiationand assigned randomly to the treatment or control group. 24 h later,mice were given one intradermal injection of 0.5×10⁶ or 1.0×10⁶ KARPAS299 cells in order to form a measurable subcutaneous tumor within 7days. Tumor size area was measured every other day. In the first set,three days after the injection of 1 million KARPAS 299 cells, 2 millionCD4CAR T (5 mice) or 2 million GFP T control cells (5 mice) wereadministered to the mice intravenously (by tail vein injection). Asecond dose of 8 million cells was injected intravenously on Day 22. Inthe second set, 10 NSG mice was irradiated and injected with 0.5×10⁶KARPAS 299 cells. On day 2, mice were injected intravenously with onecourse of 8 million CD4CAR T cells (5 mice) and 8 million GFP T controlcells (5 mice). A second dose of 5.5 million cells was injectedintravenously on Day 10. In the third set, 10 NSG mice were irradiatedand injected with 0.5×10⁶ KARPAS 299 cells. On day 1, mice wereintravenously injected with 2.5×10⁶ CD4CAR T cells or with GFP T controlcells (5 mice per group). Intravenous injections were repeated every 5days for a total of four courses.

Results Generation of the Third Generation of CD4CAR

The scFv (single-chain variable fragment) nucleotide sequence of theanti-CD4 molecule was derived from humanized monoclonal ibalizumab (alsoknown as Hu5A8 or TNX-355). This monoclonal antibody has been used in avariety of Phase I or II clinical trials. To improve signal transductionthrough the CD4CAR, the intracellular domains of CD28 and 4-1BBco-stimulators were fused to the CD3 zeta signaling domain.Additionally, the leader sequence of CD8 was introduced for efficientexpression of the CD4CAR molecule on the cell surface. Indeed, theanti-CD4 scFv is linked to the intracellular signaling domains by aCD8-derived hinge (H) and transmembrane (TM) regions (FIG. 1A). TheCD4CAR DNA molecule was sub-cloned into a lentiviral plasmid. Because ofthe presence of two co-stimulatory domains (CD28 and 4-1BB), CD4CAR isconsidered to be a third generation CAR. CD4CAR expression is controlledunder a strong SFFV (spleen focus-forming virus) promoter and is wellsuited for hematological applications.

Characterization of CD4CAR

In order to verify the CD4CAR construct, transfected 293-FT cells weresubjected to Western blot analysis. Immunoblotting with an anti-CD3zetamonoclonal antibody showed bands of predicted size for the CD4CARCD3zeta fusion protein (FIG. 1B). As expected, no CD3zeta expression wasobserved for the GFP control vector (FIG. 1B). The generated CD4CARlentiviruses were also tested for transduction efficiency in HEK293cells via flow cytometry for scFv (FIG. 6). Therefore, we confirmed thatour generated third-generation CD4CAR contained the CD3zetaintracellular domain on the intracellular end and the scFv on theextracellular end, implying that all other elements were present: CD8hinge and transmembrane domains, and CD28 and 4-1BB co-stimulatorydomains (FIG. 1C). For preclinical characterization of CD4CAR expressionand function in T cells, human T cells were activated with anti-CD3antibodies and IL-2, then transduced respectively with CD4CAR and GFPcontrol lentiviral supernatants. The T cells were then expanded for 7days after transduction.

Cord Blood-Derived CD4CAR T Cells are Highly Enriched for CD8+ T Cellsand Most of Them Bear a Central Memory T Cell Like Immunophenotype.

Human umbilical cord blood (CB) is an alternate source for allogeneic Tcell therapy. Human CB buffy coat cells were activated and transducedwith either CD4CAR or control (GFP) lentiviruses. After transduction,CD4CAR T cells and GFP T cells were expanded for 7 days, with a 20-foldincrease in cell count observed for both CD4CAR and GFP T cells (FIG.7). At day 7, cells were analyzed by flow cytometry for T-cell subsets(FIG. 2A). Flow cytometry analysis showed that ˜54% of T-cells expressedthe CD4CAR (FIG. 2B). Furthermore, we analyzed the CD4 and CD8 subsetsduring the course of T expansion following CD4CAR transduction.Consistent with previous findings, a small subset of CD8 cells wasinduced to express CD4 during T-cell activation with anti-CD3 andcostimulatory molecules (FIG. 2C). As expected, the CD4+ T subset wasalmost completely depleted within 3 or 4 days following CD4CARtransduction as compared to GFP control, in which ˜33% of cells remainedCD4+ (FIG. 2C). These data indicate that CD4CAR T cells exhibit potentanti-CD4 activity in vitro during T cell expansion.

We also evaluated the immunophenotype of CD4CAR T cells at the end ofeach culture. Following stimulation, naïve T-cells lose CD45RA and gainCD45RO in order to become central memory T-cells. Flow cytometryanalysis from 3 representative experiments showed that 96% of theexpanded T cells were CD45RO+, ˜83% were CD62L+ and ˜80% wereCD8+CD45RO+CD62L+ whereas fewer than 4% were CD45RA+ (FIG. 2D). TheCD8+CD45RO+CD62L+ immunophenotype is consistent with the acquisition ofa central memory-like phenotype, and low CD45RA+ expression confirmsloss of naïve T cell status.

CD4CAR T Cells Derived from Cord Blood Specifically Kill CD4-ExpressingLeukemia/Lymphoma Including Anaplastic Large Cell Lymphoma, SezarySyndrome and Unclassfified PTCL Lymphoma.

CD4CAR T cells highly enriched for CD8+ T cells were generated (FIG.2C). The cells were then tested in vitro for anti-leukemic functionsusing the KARPAS 299 cell line. The KARPAS 299 cell line was initiallyestablished from the peripheral blood of a patient with anaplastic largeT cell lymphoma expressing CD4. Cytogenetic analysis has previouslyshown that KARPAS 299 cells have many cytogenetic abnormalities. Duringco-culture experiments, CD4CAR cells exhibited profound leukemic cellkilling abilities (FIG. 3A). First, CB-derived CD4CAR T cells weretested for their ability to ablate KARPAS 299 cells. Indeed, at 24 hincubation and at a low E:T (effector: target) ratio of 2:1, CD4CARcells successfully eliminated KARPAS 299 cells. As a control, the CD4CART cells were also tested for their ability to ablate CD4 negativelymphoma cells. SP53 mantle cell lymphoma cell line is a human B-celllymphoma cell line that does not express CD4. Flow cytometry analysisshowed that CD4CAR T cells were unable to lyse or eliminate SP53 mantlecell lymphoma (FIG. 3D).

Studies were also conducted using patient samples. Patient 1 presentedwith an aggressive form of CD4+ T cell leukemia, Sezary syndrome, whichdid not respond to standard chemotherapy.

Patient 2 presented with an unspecified CD4+ PTCL lymphoma. Flowcytometry analysis of both patient samples revealed strong and uniformCD4 expression, with almost all leukemic cells expressing CD4 (FIGS. 3Band 3C). As visualized by flow cytometry analysis, co-culture of patientsamples with CD4CAR for 24 hours resulted in rapid and definitiveablation of CD4+ malignancies, with, once again, approximately 98%ablation observed for both Sezary syndrome and PTCL co-cultures,consistent with the ablation of KARPAS previously shown (FIGS. 3B and3C). Therefore, we show that, in a co-culture assay, CD4CAR T cellsefficiently eliminate two different types of aggressive CD4+lymphoma/leukemia cells directly from patient samples even at the lowE:T ratio of 2:1 (FIGS. 3B and 3C). These data support that CD4 is apromising therapeutic target for CD4 positive T-cell leukemias andlymphomas, analogous to the role of CD19 in the targeting of B-cellmalignancies via anti-CD19 CAR. Therefore, our patient sample and CD4CARco-culture assay extends the notion of using CAR to target CD4 positivemalignancies.

CD4CAR T Cells Derived from PBMCs Specifically Kill CD4-Expressing theTumor Cell Line.

Since autologous adoptive CAR T therapy is commonly used in the clinic,we then tested CD4CAR T cells derived from PBMCs (peripheral bloodmononuclear cells). PBMCs were activated and transduced with CD4CARlentiviruses. The CD4 and CD8 sets were monitored by flow cytometryduring cell expansion, and compared to that of cells transduced withcontrol GFP. The PBMCs derived CD4CAR T cells were highly enriched forCD8+ T cells as observed with CD4CAR T cells derived from CB (FIG. 4A),indicative of the role of CD4CAR in the depletion of CD4+. PBMC derivedCD4CAR cells were subsequently tested in their ability to ablate CD4positive leukemia/lymphoma cells, using the KARPAS 299 cell line. Theablation assay involved the co-culture of CD4CAR T cells or GFP T cells,with KARPAS 299 cells, and with the SP53 mantle cell lymphoma cell linenegative control. Reactions were stopped after 24 hours: dead cells werestained with 7-AAD (7-aminoactinomycin D) and live cells were analyzedby flow cytometry. KARPAS 299 cells incubated with CD4CAR T cellsovernight were eliminated at a rate of 38%, 62%, and 85%, at E:T ratiosof 2:1, 5:1, and 10:1, respectively (FIG. 4B). Combined, these datademonstrate a strong dose-response relationship. When target cells wereincubated with GFP control T cells, no killing of KARPAS 299 cells wasobserved. These results demonstrate that CD4CAR T cell ablation isspecific to CD4+ targeting.

CD4CAR T Cells Exhibit Significant Anti-Tumor Activity In Vivo.

In order to evaluate in vivo anti-tumor activities, we developed axenogeneic mouse model using the KARPAS 299 cell line. Multipledifferent settings were used to test CD4CAR T cell efficacy in vivo. Wefirst tested ability of the CD4CAR T cells to delay the appearance ofleukemia in the NSG mice with a single low dose. Prior to the injection,modified T cells displayed ˜40 to 50% of cells expressing CD4CAR asdemonstrated by flow cytometry analysis. Mice received intradermalinjections of KARPAS 299 cells and then a low dose (2 million) of singlesystemic injection (intravenous administration) of CD4CAR T cells wasgiven. A single low dose of systemic CD4CART cells administration toleukemia-bearing mice caused only transient regression or delayed theappearance of leukemic mass (FIG. 5A). When leukemia growth started toaccelerate, an additional course of administration of 8×10⁶ CD4CAR Tcells remarkably arrested the leukemic growth (FIG. 5A).

To further test the efficacy of CD4CAR anti-leukemia activity, weadministered two courses of relatively large doses of CD4CAR T cells.Similarly, two injections totaling 13.5×10⁶ CD4CAR T cells caused morepronounced leukemia growth arrest as compared to a lower CD4CAR dose buteventually the leukemic cell population recovered (FIG. 5B). Finally, weinvestigated the efficacy of multiple course injections of a low dose ofCD4CAR T cells (each 2.5×10⁶ cells). We treated the mice bearingsubcutaneous leukemia with repeat intravenous injections of CD4CAR Tcells, once every 4 or 5 days for total of 4 injections. After fourcourses of CD4CAR T cell administration, one of four treated mice wastumor free and exhibited no toxic appearance. Multiple dose CD4CAR Tcell-treated mice displayed more significant anti-leukemic effectcompared to single dose (FIGS. 5C and 5A). Moreover, treatment withCD4CAR T cells significantly prolonged the survival of mice bearingKARPAS 299 lymphoma as compared to treatment with the GFP-transducedcontrol T cells (FIG. 5D).

Anti-CD4 Chimeric Antigen Receptor (CD4CAR) NK Cells Efficiently TargetT-Cell Malignancies in Preclinical Models Methods Materials PrimaryTumor Cells and Cell Lines

Human leukemia cells were obtained from residual samples on a protocolapproved by the Institutional Review Board of Stony Brook University.Cord blood cells were also obtained under protocol from donors at StonyBrook University Hospital. Written, informed consent was obtained fromall donors. Karpas 299, HL-60, CCRF-CEM, MOLT4 and NK-92 cell lines wereobtained from ATCC (Manassas, Va.). NK-92 cells were cultured infiltered NK cell media, defined as alpha-MEM without ribonucleosides anddeoxyribonucleosides with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate,12.5% heat-inactivated horse serum, 12.5% heat-inactivated FBS, 1XPen/Strep, 0.2% inositol, 0.02% folic acid, and 50 μMbeta-mercaptoethanol, supplemented with IL-2 (300 IU/mL), unlessotherwise specified. Karpas 299, CCRF-CEM, and MOLT4 cell lines werecultured in RPMI, 10% FBS, 1× Pen/Strep (Gibco, Waltham, Mass., USA).HL-60 cells were cultured in IMDM, 10% FBS, 1× Pen/Strep (Gibco,Waltham, Mass., USA).

CAR Construct Generation

The CD4-specific CAR (pRSC.SFFV.CD4.3G) was designed to contain anintracellular CD28 domain upstream of 4-1BB and CD3zeta domains, therebymaking the construct a third-generation CAR.

Lentivirus Production and Transduction

To produce viral supernatant, 293FT-cells were co-transfected with pMD2Gand pSPAX viral packaging plasmids containing either pRSC.SFFV.CD4.3G orGFP lentiviral vector control, using Lipofectamine 2000 (LifeTechnologies, Carlsbad, Calif.) per manufacturer's protocol. NK cellswere cultured for a minimum of 2 days in the presence of 300 IU/mL IL-2prior to transduction with viral supernatant. Transfection andtransduction procedures are further described in Supplemental Data.

CAR Detection on Transduced NK Cells

In order to determine CAR expression, NK cells were washed and suspendedin FACs buffer (0.2% BSA in DPBS) 3 days after transduction. Normal goatIgG (Jackson Immunoresearch, West Grove, Pa.) was used to blocknonspecific binding. Each NK cell sample was probed with Biotin-labeledpolyclonal goat anti-mouse F(Ab′)² (1:250, Jackson Immunoresearch, WestGrove, Pa.) for 30 minutes at 4° C. Cells were washed once, andresuspended in FACs buffer. Cells were then stained with PE-labeledstreptavidin (1:250, Jackson Immuno Research, West Grove, Pa.) for 30minutes at 4° C. Cells were washed with FACs buffer, and resuspended in2% formalin. Flow cytometry was performed using a FACS Caliburinstrument (Becton Dickinson, Franklin Lakes, N.J.), and results wereanalyzed using Kaluza software (Beckman Coulter, Brea, Calif.).

Co-Culture Assays

CD4CAR or vector control NK cells were incubated with CD4 expressingKarpas 299 cells (anaplastic large T-cell lymphoma), HL-60 cells (acutepromyelocytic leukemia), CCRF-CEM cells (T-cell acute lymphoblasticleukemia: T-ALL), CD4⁺ T-cells isolated from human cord blood, or CD4expressing primary human leukemic cells (adult Sézary syndrome andpediatric T-ALL) at ratios of 2:1 and 5:1 (200,000 and 500,000 effectorcells to 100,000 target cells, respectively) in 1 mL of NK-cell culturemedia, without IL-2. After 24 hours of co-culture, remaining live cellswere harvested and stained with mouse anti-human CD56 and CD4antibodies, and were incubated at 4° C. for 30 minutes. CD56⁺ singlepositive denoted NK cells, and CD4⁺ single positive denoted targetcells. All cells were washed with FACs buffer, suspended in 2% formalin,and analyzed by flow cytometry.

Cytotoxicity Assay

CD4CAR or vector control NK cells were incubated with a 50:50 mix ofon-target cells (CFSE-stained Karpas 299 cells and CMTMR-stainedCCRF-CEM cells) and off-target CMTR-labelled MOLT4 cells at effector:target ratios of 1:1, 1:2, and 1:4 ratios in 1 mL of NK-cell culturemedia, without IL-2. After 24 hours, cells were stained with 7-AAD(BioLegend, San Diego, Calif.), washed with FACS buffer, and live 7-AADnegative cells were analyzed by flow cytometry.

Colony Forming Unit (CFU) Assay

CD4CAR NK cells were incubated at co-culture effector: target ratios of2:1 and 5:1 respectively with 500 CD34+ CB cells for 24 hours in NK cellmedia supplemented with IL-2. Controls used were CD34+ cells alone, andnon-transduced NK cells co-cultured at 2:1 and 5:1 effector:targetratios with CD34+ CB cells. Hematopoietic compartment output wasassessed via formation of erythroid burst-forming units (BFU-E) andnumber of granulocyte/monocyte colony-forming units (CFU-GM) at Day 16.CFU statistical analysis was performed via 2-way ANOVA with alpha set at0.05.

Xenogeneic Mouse Model

Male 12-week-old NSG mice (NOD.Cg-Prkdcsid Il2rgtm1Wjl/SzJ) werepurchased from the Jackson Laboratory (Bar Harbor, Me.) and used under aStony Brook University IACUC-approved protocol. NSG mice were irradiatedwith a sublethal (2.5 Gy) dose of gamma irradiation. Twenty-four hourslater, mice were intradermally injected with 0.5×10⁶ Karpas 299 cellsthat had been stably transduced to express luciferase, in order to causea measurable subcutaneous tumor to form. On day 1, twenty-four hoursfollowing Karpas 299 cell injection, mice were intravenously injectedvia tail vein with 5×10⁶ CD4CAR NK cells or vector control NK cells (N=4per group). Intravenous injections were repeated every 5 days for 6courses total. Tumor size area was measured every other day. On days 7,14, and 21 following Karpas 299 cell injection, mice were injectedsubcutaneously with 100 μL RediJect D-Luciferin (Perkin Elmer, Waltham,Mass.) and subjected to IVIS imaging (PerkinElmer, Waltham, Mass.).Images were analyzed using Caliper Life Sciences software (PerkinElmer,Waltham, Mass).

Statistics

Xenogeneic model sample sizes were estimated using 2-sample, 2-sidedequality power analysis (90% power and <5% significance). UnpairedStudent T tests were used to determine significance of tumor size areaand light intensity. Survival curves were constructed using theKaplan-Meier method and statistical analyses of survival was performedusing a log-rank (Mantel-Cox) test with P<0.05 considered significant.Statistical analyses were performed using GraphPad Prism 6 software.Variance was determined to be similar between the treatment and controlgroup prior to unpaired student-test.

Results Generation of the Third Generation CD4CAR

The single-chain variable fragment (scFv) nucleotide sequence of theanti-CD4 molecule was derived from the humanized monoclonal antibodyibalizumab (Hu5A8 or TNX-355)—the safety and efficacy of which have beenwell studied in clinical trials for HIV. To improve signal transduction,the CD4CAR was designed with CD28 and 4-1BB domains fused to the CD3zetasignaling domain, making it a third generation CAR. CD19-targeting thirdgeneration CAR T-cells have previously been used in clinical trials,with great efficacy. For efficient expression of the CD4CAR molecule onthe NK cell surface, a strong spleen focus-forming virus promoter (SFFV)was used and the leader sequence of CD8 was incorporated in theconstruct. The anti-CD4 scFv was separated from the intracellularsignaling domains by CD8-derived hinge (H) and transmembrane (TM)regions (FIGS. 8A and 8C). The CD4CAR DNA molecule was subsequentlysub-cloned into a lentiviral plasmid.

Characterization of CD4CAR

In order to validate the CD4CAR construct, HEK293-FT cells weretransfected with the CD4CAR lentiviral plasmid or vector controlplasmid, and 48 hours later were harvested for Western blot analysis.Immunoblotting with an anti-CD3zeta monoclonal antibody showed bands ofpredicted size for the CD4CAR-CD3zeta fusion protein (FIG. 8B). Asexpected, no CD3zeta expression was observed for the GFP vector controlprotein (FIG. 8B).

Generation of CD4CAR NK Cells

CD4CAR NK transduction efficiency was determined to be 15.9%, asdetermined by flow cytometry (FIG. 9A upper panel). Next,fluorescence-activated cell sorting (FACS) was used in order to furtherenrich for CD4CAR⁺ NK cells. Following sorting, collected CD4CAR^(high)NK cells were confirmed to be more than 85% CD4CAR positive (FIG. 15).After FACS collection of CD4CAR^(high) cells, CD4CAR expression levelsremained consistently stable at 75-90% on NK cells during expansion ofup to 10 passages, and following cryopreservation. Indeed, at the onsetof co-culture experiments, expanded CD4CAR^(high) NK cells expressed CARat 85% (FIG. 9A lower panel).

CD4CAR NK Cells Specifically Lyse CD4⁺ Blood Cancer Cells IncludingAnaplastic Large T-Cell Lymphoma (Karpas 299), Acute Myeloid Leukemia(HL-60) and T-Cell Acute Lymphoblastic Leukemia (CCRF-CEM)

CD4CAR NK cells were tested for anti-lymphoma activity in vitro usingthe following CD4⁺ cell lines: Karpas 299, HL-60, and CCRF-CEM. TheKarpas 299 cell line was established from the peripheral blood of a25-year-old patient with anaplastic large T-cell lymphoma. The HL-60cell line was established from the peripheral blood of a 36-year-oldpatient with acute promyelocytic leukemia. The CCRF-CEM cell line wasestablished from the peripheral blood of a 4-year-old patient withT-cell acute lymphoblastic leukemia (T-ALL).

During 24-hour co-culture experiments, CD4CAR NK cells showed profoundkilling of CD4 positive leukemia/lymphoma cells at the low effector cellto target cell ratio (E:T) of 2:1 (FIG. 10A) and the standard 5:1 ratio(FIG. 10C). In co-culture cytotoxicity assays, target tumor cells wereidentified by the CD4⁺, CD56⁻ immunophenotype (labeled in blue on flowcytometry charts). As expected, vector control NK cells showed somenon-specific tumor cell killing ability that is innate to NK cells, butas expected, were far less effective against CD4⁺ tumor cells comparedto CD4CAR NK cells. Analysis of Karpas 299 cells alone confirmed 99.1%CD4⁺ expression (FIG. 1A upper panel). Strikingly, at an E:T ratio of2:1, CD4CAR NK cells completely ablated 100% of Karpas 299 cellscompared to vector control (N=2) (FIGS. 10A upper panel and 10C).Similarly, analysis of HL-60 and CCRF-CEM cells alone confirmed highexpression of CD4, 99.9% and 92.1%, respectively (FIG. 10A middle andlower panels). Likewise, at an E:T ratio of 2:1, CD4CAR NK cellsrobustly lysed 75% of HL-60 cells and 97% of CCRF-CEM cells, as comparedto vector control (FIGS. 10A and 10C). Combined, these data show thatCD4CAR NK cells specifically and potently target CD4⁺ cells in additionto retaining non-specific anti-tumor cell activity intrinsic to NKcells.

Co-culture studies were also conducted using patient samples (FIGS. 10Band 10C). Patient 1 presented with Sézary syndrome, an aggressive formof CD4⁺ cutaneous T-cell lymphoma that did not respond to standardchemotherapy. Sézary syndrome is a subset of PTCL. Patient 1's leukemiccells were assessed to be 78.1% CD4⁺ via flow cytometry (FIG. 10B).Patient 2 presented with a CD4⁺ pediatric T-cell acute lymphoblasticleukemia (T-ALL). Analogously, Patient 2's cells were assessed to be43.7% CD4⁺ via flow cytometry (FIG. 10B). After 24 hours of co-cultureat a low E:T ratio of 2:1, CD4CAR NK cells lysed 58% of CD4⁺ Sézarysyndrome cells from patient 1, and 78% of CD4⁺ T-ALL cells from patient2 (N=2). Furthermore, at an increased E:T ratio of 5:1, standard for CARco-culture assays, CD4CAR NK cells lysed 82% of Sézary syndrome cellsfrom patient 1, and 82% of T-ALL cells from patient 2 (N=2) (FIG. 10Cand FIG. 14). These data strongly suggest a dose-dependent response andpotent CD4CAR NK cell anti-tumor activity in a cell line and patientsample setting for both adult and pediatric CD4⁺ T cell leukemias andlymphomas.

CD4CAR NK Cells Specifically Lyse CD4-Expressing Tumor Cell Lines inDose Dependent Manner.

CD4CAR NK cells specifically lyse CD4⁺ Karpas 299 and CCRF-CEM leukemiccell lines in vitro in a dose-dependent manner at effector: targetratios of 1:4, 1:2, and 1:1 (FIG. 11). For each co-culture E:T ratio,CD4CAR NK effector cells or vector control NK effector cells wereincubated with tumor cells that were comprised of equal numbers ofon-target CD4⁺ cells, CFSE-stained Karpas 299 or CFSE-stained CCRF-CEM,and “off-target” CMTMR-stained CD4⁻, CD5⁺ MOLT4 acute lymphoblasticleukemia cells. The MOLT4 cells were included to account for variationin the starting cell numbers and for spontaneous target cell death.After 24 hours, live cells were analyzed by flow cytometry. Percentlysis of target cells was measured by comparing CD4⁺ target cellsurvival in CD4CAR NK co-culture to vector control NK co-culture. Karpas299 cells were eliminated at rates of 67%, 95%, and 100%, at effector totarget ratios of 1:4, 1:2, and 1:1, respectively (FIG. 11). And CCRF-CEMcells were eliminated at rates of 39%, 58%, and 69% respectively at thesame E:T ratios (FIG. 11). As expected, CD4CAR NK cells did not lyseCMTMR-labeled MOLT4 cells, confirmed to be <5% CD4⁺ by flow cytometryanalysis (FIG. 16A). Additional co-culture experiments confirmed thatCD4CAR NK cells did not lyse MOLT4 cells at 0 h, 4 h, 8 h, and 24 h(FIG. 16B), whereas CD4CAR NK cells lysed Karpas 299 cells as detectedby flow cytometry as early as 4 h (FIG. 16C). Combined, these dataindicate that CD4CAR NK cell anti-tumor cytotoxicity is dose-dependent,rapid onset and highly specific to CD4⁺ cells.

Additional co-culture studies were conducted using CD4⁺ T-cells isolatedfrom cord blood. In these experiments, CD4CAR NK cells completelydepleted CD4⁺ T-cells at an effector:target ratio of 2:1 after 24 hoursof co-culture, with remaining cells 0.0% CD4⁺. As expected, after CD4⁺cord blood cell co-culture with corresponding vector control NK cells(CD56⁺, CD4⁻), the CD4⁺ population remained largely intact (FIG. 12A),further confirming specific and robust CD4CAR NK-mediated depletion ofCD4⁺ populations on healthy tissue. CD4CAR NK cells do not affect stemcell output in hematopoietic compartment.

CFU (Colony-Forming-Unit) assay analysis revealed that CD4CAR NK cellsdid not significantly affect the CD34+ cord blood stem cell output ofthe hematopoietic compartment. Hematopoietic compartment output wasassessed by the presence of erythroid progenitors andgranulocyte/macrophage progenitors at Day 0, determined by number oferythroid burst-forming units (BFU-E) and number of granulocyte/monocytecolony-forming units (CFU-GM) at Day 16 (FIG. 12B). This finding isconsistent with specific targeting of CD4, a mature T-cell marker, withlimited impact on hematopoietic stem cells and early progenitors, and noevidence of lineage skewing, a measure of therapeutic safety.

CD4CAR NK Cells Exhibit Significant Anti-Tumor Activity In Vivo

In order to evaluate the in vivo anti-tumor activity of CD4CAR NK cells,we developed a xenogeneic mouse model using NSG mice sublethallyirradiated and intradermally injected with luciferase-expressing Karpas299 cells to induce measurable tumor formation. On day 1, 24 hoursfollowing Karpas 299 cell injection, and every 5 days afterwards for atotal of 6 courses, mice were intravenously injected with 5×10⁶ CD4CARNK cells or vector control NK control cells per administration. On days7, 14, and 21, mice were injected subcutaneously with RediJectD-Luciferin and underwent IVIS imaging to measure tumor burden (FIG.13A). Average light intensity measured for the CD4CAR NK injected micewas compared to that of vector control NK injected mice (FIG. 13B). ByDay 21, the CD4CAR NK injected mice had significantly less lightintensity and therefore thus less tumor burden compared to vectorcontrol (p<0.01). On day 1, and every other day afterwards, tumor sizearea was measured and the average tumor size between the two groups wascompared (FIG. 13C). Unpaired student T test analysis revealed that theaverage tumor size of CD4CAR NK injected mice was significantly smallerthan that of vector control NK injected mice starting on day 17 (p<0.05)and continuing on days 19-25 (p<0.01). Next, we compared mouse survivalacross the two groups (FIG. 13D). All of the CD4CAR NK injected micesurvived past day 30. However, percent survival of vector control NKinjected mice started to decrease on day 17 with no survival by day 23.In summary, these in vivo data indicate that CD4CAR NK cellssignificantly reduce tumor burden and prolong survival in Karpas299-injected NSG mice.

Anti-CD5 Chimeric Antigen Receptor (CD5CAR) T Cells Efficiently TargetCD5 Positive Hematologic Malignancies Examples Results Generation of theThird Generation of CD5CAR

The construct for CD5CAR, as well as anchored CD5 scFv antibody weredesigned to test the function and mechanism of CD5CAR T cells in termsof both the targeting and lysis of CD5 expressing cells and the abilityof CD5CAR T cells to down-regulate CD5 expression within their ownCD5CAR T-cell population (FIG. 17A). To confirm the CD5CAR construct,the generated CD5CAR lentiviruses were transduced into HEK293 cells.After 48 h treatment with CD5CAR or GFP-lentiviruses, the expression ofCD5CAR in HEK293 cells was verified by Western blot analysis usingCD3zeta antibody, which recognize C-terminal region of CD5CAR protein(FIG. 17B). The resulting band was the predicted size of CD5CAR proteinin CD5CAR transduced HEK293 cells, but GFP transduced HEK293 cells didnot exhibit any specific band by Western blot analysis. In order toevaluate the function of CD5CAR protein for future experiments, CD5CARlentiviruses were transduced into activated human T cells. Theexpression of CD5CAR on surface of T cells was evaluated by flowcytometry analysis using goat anti-mouse F(ab′) antibody, whichrecognizes scFv region of CD5CAR protein. Flow cytometric analysisshowed that about 20% of CD5CAR expression was observed on CD5CARtransduced T-cells compared to isotype control (FIG. 17C). These resultsindicated that we successfully generated CD5CAR expression T cell forfollowing experiments.

Down-Regulation of CD5 Expression for CAR Therapy

Prior to CD5CAR T cell co-culture and animal assays, the expression ofCD5 on the surface of CD5CAR T cells is down regulated to avoidself-killing within the CD5CAR T population. The down-regulation of CD5will prevent the self-killing of CAR T cells within the CAR T cellpopulation, and the down-regulation of CD5 is associated with anincreased killing ability of T-cells. A CAR that is produced withinT-cells that has no CD5 expression could be a super-functional CAR, nomatter the construct of the CAR itself. The steps for generation of CD5CAR T cells and the comparison of CD5 down-regulation using single ordouble transduction of CD5 CAR lentiviuses are shown in FIGS. 18A and B.The single transduced CD5CAR T cells with un-concentrated lent-CD5 CARviruses did not show complete downregulation of CD5 protein from cellsurface by day 8, with a maximum CD5 negative population up to 46% onday 6 (FIG. 18C). In the double transduced population, about 90% oftransduced T cells became CD5 negative on day 4-day incubation. Incontrast, the GFP T-cell control maintains a CD5+, CD3+ double positivepopulation above 95% from day 2 through day 8 (FIG. 18C).

Downregulation of CD5 Expression on T-Cells can be Accomplished byTransduction of Anchored CD5CAR scFv Lentiviruses.

In order to further elucidate the mechanism by which CD5CARdown-regulates CD5 expression on T cells, a new construct was createdentitled anchored CD5 scFv (SEQ ID NO. 7 (FIG. 17A). This constructincludes an anti-CD5 scFv lined to a transmembrane domain via a hingeregion, which allows CD5 scFv to anchor on the T cell surface. Theanchored CD5 scFv polypeptide (SEQ ID NO. 16) binds to CD5 targetwithout target cell lysis as observed with a functional CD5CAR. A singletransduction and flow data analysis is shown in FIGS. 19A and 19B, withpartial down-regulation of CD5 expression for T cells on day 7 ofincubation. This is consistent with the partial down-regulation of CD5expression seen for CD5CAR T-cells after a single transduction.

CD5CAR T Cells Effectively Lyse T-Cell ALL Cell Lines.

The killing ability of CD5CAR T cells was first tested against T-cellALL established cell lines CCRF-CEM and MOLT-4, and an anaplastic largecell leukemic cell line KARPAS 299 as shown in FIGS. 20A and 20B. Anavid killing ability was seen for the two CD5+ cell lines when comparedto GFP control, with target cell lysis above 75% for both lines. 0%lysis was observed in an analplastic large cell line KARPAS 299, whichis negative for CD5.

CD5CAR T Cells Effectively Lyse T-Cell ALL Cells from Human Samples.

The CD5CAR ability to lyse patient sample T-ALL cells was also assessedusing multiple patient samples and CD5CAR cell co-cultures were shown inFIG. 21 and FIG. 22. While there was an avid cell killing noted for theT-ALL 1 patient leukemic cells that was similar to the CD5 target celllysis seen when CD5CAR cells targeted T cell ALL cell lines, three otherpatient leukemic cells showed comparatively weaker lysis of target cells(FIG. 21A. and FIG. 21B.).

The ability of killing by CD5CAR on the patient leukemic cellscorrelated with the intensity of CD5 expression as shown in FIGS. 21A,21B, and 21D. As shown in FIGS. 21C, and 21D, the CD5 expression forT-ALL-1, T-ALL 3, T-ALL 6 and T-ALL 7 through flow cytometry analysiswas observed. The CD5 expression was significantly lower for the T-ALLpatient samples, except for T-ALL-1 sample.

CD5CAR T Cells Exhibit the Specificity and Potent Target Cell Killing.

As a control, the CD5CAR T cells were also tested for their ability toablate CD5 negative leukemic T cells. Anaplastic large T cell lymphomaline is the cell line that does not express CD5. Flow cytometry analysisshowed that CD5CAR T cells were unable to lyse or eliminate KARPAS 299cells, as shown in FIG. 21A, lower panel.

A patient sample (T-ALL-8) with a high level of CD5 expression wasobtained from a patient with a minimal disease of T-ALL. Co-culture wasperformed with CD5CAR and analyzed in detail as shown in FIG. 22. Threepopulation cells including CD5+ normal T cells, CD5+CD34+ T-ALL cellsand CD5-CD34+ T-ALL cells were assessed by flow cytometry afterco-culture. CD5CAR exhibited the specificity and potent target celllysis ability with >93% of CD5 positive cell lysis for all CD5+ cellpopulations when compared to GFP control. The CD5CAR killed leukemiccells as efficiently as CD5 normal T cells. Killing was not observed inthe CD5 negative population. CD5CAR T cells essentially eliminated the Tcell population (CD5+CD34−).

CD5CAR T Cells Effectively Eliminate Normal T Cells.

CD5CAR T cells demonstrated effective elimination of normal T cells in adose dependent manner in a co-culture assay at low ratios(effector:target) of 0.25:1, 0.5:1 and 1:1 (FIG. 23). CD5CAR T cells orCD123CAR T (control) effector cells were incubated with GFP labeled Tcells. Percent killing of target cells was measured by comparing GFP Tcell survival in CD5CAR T co-culture relative to that in CD123CAR Tcontrol co-culture. Normal GFP T cells were eliminated in adose-response fashion for CD5CAR T cells. CD5CAR T cells effectivelyeliminated all GFP T cells at effector to target ratio of 1:1 (FIG. 23).Since the CD5CAR T cells effectively eliminated all normal T cells, thefeasibility of CD5CAR T therapy should depend on the ability to providetransient rather than permanent. CD5CAR T cells could be used as a novelconditioning regimen or a “bridge” for hematopoietic celltransplantation.

T Cells Maintained CD5 Expression When They Were Co-Cultured with CD5CARor Anchored CD5 scFv T Cells.

One of CD5 properties is its internalization after binding by anantibody. As a result, targeting cells lose a targeted antigen, whichmay cause an antigen escape. This phenomenon has been reported as acause of failure in clinical studies using CAR T-cell based therapies.We next investigate the issue if CD5 CAR or anchored CD5 scFv T cellsaffect the CD5 expression on CD5 positive T or leukemic cells using aco-culture assay. Steps for generation of CD5CAR T cells or anchored CD5scFv T cells and CD123 CAR T cells (control) were shown in FIG. 24A.After the second T cell transduction with lenti-CD5CAR or anchored CD5scFv and CD123CAR viruses on day 3, transduced T cells were analyzedwith the expression of CD5 by flow cytometry. T cells transduced eitherCD5CAR or anchored CD5 scFv lentiviruses displayed essentially completedownregulation of the surface CD5 protein (FIG. 24B). In contrast, theCD123 CAR transduced-T cell control maintained the CD5 expression.

We then co-cultured transduced CD5CAR or CD5 anchored scFv and CD123CART cells with GFP-labeled T cells at the ratio of 1:1 (E:T) for 2 or 4days. As shown in FIGS. 25A and 25B, CD5CAR T cells effectivelyeliminated all GFP-T cells. As expected, transduced CD5 anchored scFv orCD123CAR T cells were unable to lyse GFP T cells. In addition, GFP Tcells still expressed CD5 when co-cultured with transduced CD5 anchoredscFv or CD123CAR T cells. These studies indicate that CD5 antigen escapeis unlikely to occur when employing CD5CAR for immunotherapy.

Down-Regulation of CD5 Expression in the T ALL Cells When They WereTransduced With Lenti-CD5CAR or CD5 Anchored scFv Viruses.

We next tested if transduction of CD5CAR− or anchored CD5CARlentiviruses on T ALL cells results in the downregulation of CD5expression. CCRF-CEM and MOLT-4 T-ALL cells were transduced with CD5CAR−or anchored CD5 scFv lentiviruses. CD5CAR or anchored CD5 scFvsignificantly down-regulated or reduced the quantity of surface CD5expression on these leukemic cells (FIG. 25C). In contrast, the T cellsmaintained CD5 expression when these cells were used to co-culture withtransduced anchored CD5 scFv T cells (FIGS. 24A and 24B).

CD5CAR T Cells Exhibit Profound Anti-Tumor Activity In Vivo.

In order to evaluate the in vivo anti-tumor activity of CD5CAR T cellsas a predictor of their therapeutic efficacy in patients, we developed axenograft mouse model using NSG mice sublethally (2.0 Gy) irradiated andintravenously injected with 1.0×10⁶ firefly luciferase-expressingCCRF-CEM cells (CD5+) to induce measurable tumor formation. On day 3days following CCRF-CEM-Luc+ cell injection, mice were intravenouslyinjected with 5×10⁶ CD5CAR T cells or vector control T cells. Theseinjections were repeated on Day 4, Day 6, and Day 7, for a total of20×10⁶ T cells per mouse. On days 5, 8, 10 and 13, mice were injectedsubcutaneously with RediJect D-Luciferin (Perkin-Elmer) and subjected toIVIS imaging (Caliper LifeSciences) to measure tumor burden (FIG. 26A).Average light intensity measured for the CD5CAR T cell injected mice wascompared to that of vector control T injected mice (FIG. 26B). Paired Ttest analysis revealed a very highly significant difference between thetwo groups by day 13 with less light intensity and thus less tumorburden in the CD5CAR T injected group compared to control (p<0.0012).Further analysis showed that by Day 5, mice treated with CD5CAR T cellsonly 3 days previously had 53% lower tumor burden compared to controlmice, and that percentage improved to 95% by Day 8 (FIG. 26C.) Tumorburden remained at near background levels for treated mice through Day13. On Day 15, a small amount of peripheral blood was drawn from eachmouse including 2 mice which were not injected with wither CCRF-CEM or Tcells (to serve as background controls), and analyzed by flow cytometryfor the presence of transplanted CCRF-CEM cells (CD5+). Results mirroredthe imaging perfectly as percentage of tumor cells in CD5CAR Tcell-treated mice dropped to near background levels (<1%), while micegiven control T cells had between 28-43% CCRF-CEM tumor cells (FIG.26D). In summary, these in vivo data indicate that CD5CAR T cellsrobustly reduce tumor burden and prolong survival in CCRF-CEM-injectedNSG mice when compared to vector control T cells.

Anti-CD5 Chimeric Antigen Receptor (CD5CAR) NK Cells EfficientlyEliminate CD5 Positive Hematologic Malignancies. Examples ResultsGeneration of the CD5NK-CAR

The anti-CD5 molecule is a modular design, comprising of a single-chainvariable fragment (scFv) in conjunction with CD28 and 4-1BB domainsfused to the CD3zeta signaling domain to improve signal transductionmaking it a third generation CAR. A strong spleen focus forming viruspromoter (SFFV) was used for efficient expression of the CD5CAR moleculeon the NK cell surface and the CD8 leader sequence was incorporated intothe construct. The anti-CD5 scFv is attached to the intracellularsignaling domains via a CD8-derived hinge (H) and transmembrane (TM)regions. This CD5CAR construct was then cloned into a lentiviralplasmid.

Generation of CD5CAR NK Cells

The transduction efficiency of the CD5CAR was determined by flowcytometry analysis. To enrich for CD5CAR+ NK cells, the highestexpressing NK cells were harvested using flow cytometry. Followingsorting, the expression of the CD5CAR^(high) NK was expanded forefficacy studies in vitro and vivo.

CD5CAR NK Cells Effectively Eliminate Human T-Cell Acute LymphomblasticLeukemia (T-ALL) Cell Lines

CD5CAR NK cells were tested for anti-T-ALL activity in vitro usingCCRF-CEM, MOLT-4 and Jurkat cell lines. All these T-ALL cell lineshighly expressed CD5.

During co-culture experiments, CD5CAR NK cells demonstrated profoundkilling of CCRF-CEM at the low effector cell to target cell ratio (E:T)of 2:1 and 5:1. At these ratios, CD5CAR NK cells virtually eliminatedCCRF-CEM cells (FIG. 27A). CD5CAR NK cells lysed CCRF-CEM leukemic cellsin vitro in a dose-dependent manner at effector: target ratios of0.25:1, 0.5:1, 1:1, 2:1 and 5:1 (FIGS. 27B and 27C). Additional twoT-ALL cells, MOLT-4 and Jurkat were used to test the anti-leukemicactivity for CD5NK cells. Co-culture studies of these two cell lineswere conducted with CD5CAR NK cells. CD5CAR NK cells essentiallyeliminated MOLT-4 and Jurkat cells at a low effector: target ratio of2:1 (FIGS. 28A and 28B).

CD5CAR NK Cells Effectively Eliminate Aggressive CD5+ T-ALL Cells UsingHuman Samples

Co-culture experiments were also conducted using patient samples (FIGS.29A, 29B). Both patient 1 and 2 were T-ALL that did not respond tostandard chemotherapy. Patient 1 (T-ALL #1) had a small subset of T-ALLcells positive for CD5. Leukemic cells from this patient wereco-cultured with CD5CAR NK cells. Target populations were gated andquantified with flow cytometry using cell cytotracker dye (CMTMR) tolabel patient's cells. Target CD5+CD34+ cell populations were gatedagainst an isotype control. CD5CAR NK cells lysed about 60% of CD34+CD5+leukemic cells at an E:T ratio of 5:1. Importantly, CD5CAR NK cellsshowed no any activity against CD5− cell populations, implying specificand directed activity against (selective for) target antigen epitopes.Patient 2 had a T-ALL population, which was virtually positive for CD5,and co-cultured with CD5CAR NK cells. CD5CAR NK cells showed almostcomplete lysis of the highly expressing CD5+ target population withpotent activity against the dim CD5+CD34+ population (FIG. 29B).

CD5CAR NK Cells Effectively Eliminate Aggressive CD5+ Peripheral T CellLymphoma (PTCL) Cells Using Human Samples.

Patient 3 presented a CD4+ PTCL (unclassified type) and patient 4presented with Sézary syndrome, an aggressive form of PTCLs that did notrespond to a standard chemotherapy. Lymphoma cells from patient 3 wereco-cultured with CD5CAR NK cells for 24 hours. Leukemic cells wereCD5+CD7− positive and the CD5+CD7− population was gated and quantifiedby flow cytometry. Target CD5+CD7− population was analyzed and cellsurvival was expressed relative to transduced vector control NK cells.CD5CAR NK displayed almost complete lysis of the leukemic CD5+CD7−target population, with complete lysis across the entire CD5+ populationincluding normal T cells expressing CD5 (FIG. 29C).

Leukemic cells from patient #4 with Sézary syndrome were co-culturedwith CD5CAR NK cells at E:T ratios of 2:1 and 5:1 after 24 hours. CD5CARNK cells demonstrated a potent anti-leukemic acidity with over 90% lysisof Sézary syndrome cells (FIG. 29D). Saturation was achieved at 2:1 E:Tratio where leukemic cells were virtually eliminated.

CD5CAR NK Cells Effectively Deplete Normal T Cells.

T cells were isolated from cord blood and used to co-culture with CD5CARNK cells. As shown in FIG. 30, CD5CAR NK cells completely depleted Tcells at a low effector:target ratio of 2:1 after 24 hours of co-culture(FIG. 30). As compared to that of the GFP control, the T cell populationremained largely intact.

CD5CAR NK Cells Effectively Lyse CD5+ B-Cell Malignancies IncludingMantle Cell Lymphoma (MCL) and Chronic Lymphocytic Lymphoma (CLL).

Additional co-culture studies were conducted with CD5+ Jeko lymphomacell line and lymphoma cells from patients with (MCL) and CLL. TheJeKo-1 MCL cell line was established from peripheral blood mononuclearcells of a patient with a large cell variant of MCL. In co-culturestudies at a low E:T of 2:1, CD5CAR NK cells effectively lysedapproximately 80% of Jeko cells (FIG. 31A). Cells isolated from apatient samples with MCL was also co-cultured with CD5CAR NK cells.Target populations were gated and live cells were quantified by flowcytometry. CD5CAR NK cells virtually eliminated both populations, whichwere CD5+CD19+ leukemia population and CD5+CD19− T-cell population (FIG.31B). Cells from a patient with B-cell CLL were also co-cultured withCD5CAR NK cells. CD19 was used to gate the leukemic population with flowcytometry. CD5+CD19+ CLL cells were virtually eliminated by CD5CAR NKcells (FIG. 31C). These studies strongly suggest that CD5CAR NK cellsinclude a biologcal property of profound anti-tumor activity in leukemiccell lines and patient leukemic samples (FIG. 32) including for T-ALL,PTCLs and B-cell lymphomas expressing CD5.

CD5CAR NK Cells Demonstrate a Potent Anti-Leukemic Activity In Vivo.

A similar strategy for CD5CAR T cells, animal studies were employed todetermine the in vivo anti-tumor activity of CD5CAR NK cells.Sublethally irradiated NSG mice were intravenously injected with 1.0×10⁶firefly luciferase-expressing CCRF-CEM cells to induce measurable tumorformation. 3 days following CCRF-CEM-Luc+ cell injection, mice wereintravenously injected with 5×10⁶ CD5CAR NK cells or vector control Tcells. These injections were repeated on Day 4 for a total of 10×10⁶ Tcells per mouse. On day 5, mice were injected subcutaneously withRediJect D-Luciferin and subjected to IVIS imaging to measure tumorburden (FIG. 33A). Average light intensity measured for the CD5CAR NKcell injected mice was compared to that of vector control NK cellinjected mice (FIG. 33B). Tumor burden was two thirds lower for treatedmice on day 5 after tumor injection. Paired T test analysis revealed avery highly significant difference (P=0.0302) between the two groups.These in vivo data indicate that CD5CAR NK cells significantly reducetumor burden in CCRF-CEM-injected NSG mice in a rapid manner whencompared to vector control NK cells.

Anti-CD3 Chimeric Antigen Receptor (CD3CAR) NK Cells Efficiently LyseCD3 Positive Hematologic Malignancies Examples Results Generation of theCD3CAR

The anti-CD3 molecule is a modular design, comprising of a single-chainvariable fragment (scFv) in conjunction with CD28 and 4-1BB domainsfused to the CD3zeta signaling domain to improve signal transductionmaking it a third generation CAR. A strong spleen focus forming viruspromoter (SFFV) was used for efficient expression of the CD3CAR moleculeon the NK cell (NK-92) surface and the CD8 leader sequence wasincorporated into the construct. The anti-CD3 scFv is attached to theintracellular signaling domains via a CD8-derived hinge (H) andtransmembrane (TM) regions (FIG. 34A). This CD3CAR construct was thencloned into a lentiviral plasmid.

Characterization of CD3CAR

Western blot analysis was performed on HEK293-FT cells transfected withCD3CAR lentiviral plasmid and vector control plasmid. Immunoblots withanti-CD3zeta monoclonal antibody show bands of predicted size for theCD3CAR-CD3zeta fusion protein (FIG. 34B) versus no bands for the vectorcontrol protein.

Generation of CD3CAR NK Cells Using NK-92 Cells

The transduction efficiency of the CD3CAR was determined by flowcytometry analysis. To enrich for CD3CAR NK cells, the highestexpressing NK cells were harvested using fluorescence-activated cellsorting (FACS). Following sorting, NK cells with relatively highexpression of CD3CAR was obtained. Expression of CD3CAR following flowcytometry sorting was stable around 30% of CAR expression for subsequentNK cell expansion and cryopreservation.

CD3CAR NK Cells Effectively Lyse Human T-ALL Cell Lines

To determine the efficacy for CD3CAR NK cells, we conducted co-cultureassays using CD3+ T-ALL cell lines, Jurkat, and CCRF-CEM. CD3 positivecells in Jurkat and CCRF-CEM cells are approximately 80% and 10%positive for CD3, respectively. CD3+ cells from the CCRF-CEM cell linewere then sorted for highly expressed CD3 cells, and CD3 expression insorted CCRF-CEM cells were about 50%. During co-culture with Jurkat andCCRF-CEM cells, CD3CAR NK cells demonstrated profound leukemic cellkilling abilities (FIGS. 35A-35B). At 6 hour incubation and at a low E:Tratio of 2:1, CD3CAR NK cells effectively lysed over 60% of Jurkat cells(FIG. 35A). We next compared the killing ability of relative highlyexpressed CD3 CCRF-CEM cells (sorted) with that of unsorted CCRF-CEMcells. The CD3 CAR NK cells appeared to be more efficacious against ahigher CD3 expressing population in sorted CCRF-CEM than a lower CD3expressing unsorted CCRF-CEM (FIG. 35B) population.

CD3CAR NK Cells Effectively Eliminate CD3+ Leukemic Cells From HumanSamples

The killing ability of CD3CAR NK cells was also tested using patientsamples. Flow cytometry analysis of both patient samples revealed strongand uniform CD3 expression. As analyzed by flow cytometry, co-culture ofSezary syndrome patient sample with CD3CAR T cells effectively resultedin lysis of approximately 80% of leukemic cells at a low E:T ratio of2:1 (FIG. 36A). Co-culture of patient sample, unclassified PTCLs withCD3CAR NK cells for 24 hours resulted in virtual ablation of CD3+malignant cells (FIG. 36B). The CD3CAR NK cells also affected the broadCD3+ population.

CD3CAR NK Cells are Able to Deplete Normal T Cells.

GFP transduced normal T cells were used to co-culture CD3CAR NK cells.As shown in FIG. 37, CD3CAR NK cells depleted a substantial portion ofnormal T cells after 4 or 24-hour incubation.

CD3CAR NK Cells Exhibit Profound Anti-Leukemic Activity In Vivo

To determine the in vivo anti-tumor efficacy of CD3CAR NK cells,sublethally irradiated NSG mice were intravenously injected with 1.0×10⁶firefly luciferase-expressing Jurkat cells, which are CD3 positive(˜80%), and measurable tumor formation was detected by Day 3 or 4. Threedays following Jurkat-Luc+ cell injection, mice were intravenouslyinjected with 5×10⁶ CD3CAR NK cells or vector control NK cells permouse, 6 per group. These injections were repeated on Day 3, 6, 7 and 10for a total of 25×10⁶ T cells per mouse. On days 4, 7, 9 and 13 micewere subjected to IVIS imaging to measure tumor burden (FIG. 38A). Twotreated mice died due to injection procedure on day 13. Average lightintensity measured for the CD3CAR NK cell injected mice was compared tothat of vector control NK injected mice (FIG. 38B). After an initial lagperiod, tumor burden then dropped to approximately two-thirds lower fortreated mice by Day 9 and just 13% on Day 13 (FIG. 38C). Paired T testanalysis revealed a highly significant difference (P=0.0137) between thetwo groups. We conclude that these in vivo data demonstrate that CD3CARNK cells significantly reduce tumor burden and prolong survival inJurkat-injected NSG mice when compared to vector control NK cells.

CRISPR/Cas Nucleases Target to CD2, CD3, CD5 and CD7 Expressed on T orNK Cells.

T or NK cells appear to share some of surface antigens, such as CD2,CD3, CD5 and CD7 with leukemia or lymphoma. CD2, CD3, CD5, and CD7 couldbe good targets for T and NK cells as they are expressed in most of Tcell leukemia/lymphoma.

Therefore, when one of surface antigens, CD2, CD3, CD5, and CD7 isselected as a target, this antigen is needed to delete or down-regulatein T or NK cells used to generate CAR if they share this antigen, toavoid self-killing within the CAR T or NK cell population.

Steps for generation of CAR T or NK cell targeting T-cell lymphomas orT-cell leukemia are described in FIG. 39. Three pairs of sgRNA weredesigned with CHOPCHOP to target CD2, CD3, CD5, and CD7. Gene-specificsgRNAs (FIG. 40) were then cloned into the lentiviral vector (LentiU6-sgRNA-SFFV-Cas9-puro-wpre) expressing a human Cas9 and puromycinresistance genes linked with an E2A self-cleaving linker. The U6-sgRNAcassette is in front of the Cas9 element. The expression of sgRNA andCas9puro is driven by the U6 promoter and SFFV promoter, respectively.

Examples Results CRISPR/Cas Nucleases Target to CD5 on T Cell Lines.

Lentiviruses carried gene-specific sgRNAs were used to transduceCCRF-CEM and MOLT cells. Initially, the loss of CD5 expression wasobserved in both of these T cell lines using two different twoCDISPR/Cas9 sgRNA sequences (FIGS. 41A and 41C). The most successfulpopulation in terms of the loss of CD5 expression was chosen for eachcell line, and these cells were sorted, expanded normally and found tobe of >99% purity CD45+ and CD5− (FIGS. 41B and 41D).

CRISPR/Cas Nucleases Target to CD7 on T Cell Lines and NK Cells.

Lentiviruses carried gene-specific sgRNAs were used to transduceCCRF-CEM, MOLT cells and NK cells (FIG. 42). Flow cytometry analysisdemonstrated the loss of CD7 expression in CCRF-CEM and NK-92 cells withCRISPR/Cas9 approach using two different sgRNAs (FIGS. 42A and 42B). Thepopulation (denoted by the blue circle and arrow) was selected forsorting, expansion and analysis in FIG. 42B. The loss of CD5 expressionby flow cytometry analysis was also seen in NK-92 cells using a similarapproach described above with CRISPR/Cas nucleases targeting to CD7(FIGS. 42C and 42D) The sorted CD7 negative NK-92 cells (FIG. 42D) wereexpanded and used to generate CD7CAR NK cells to eliminate CD7 pos-itiveleukemic cells.

CD7CAR NK⁷⁻-92 Cells Have a Robust Anti-Leukemic Activity

CD7 is expressed in both NK and T-ALL leukemic cells. To avoidself-killing within the CD7CAR NK-92 population, CD7 expression firstneeds to be inactivated. CD7 deficient NK-92 cells (NK⁷⁻-92 cells) weregenerated as described in (FIG. 42D) and expanded. The expanded NK⁷⁻-92cells were transduced with lentivirus expressing a CD7CAR. CD7CARincludes an anti-CD7 scFV in conjunction with CD28 and 4-BB domainsfused to CD3zeta signaling domain making it a third generation CAR.CD7CAR NK⁷⁻-92 cells were used to test their lysis ability of leukemiccells expressing CD7. As shown in FIG. 43, CD7CAR NK⁷⁻-92 cellsdisplayed a potent anti-leukemic activity against a T-ALL cell line,CCRF-CEM. As analyzed by flow cytometry, co-culture of CCRF-CEM cellseffectively resulted in the lysis of approximately 50% of leukemic cellsat E:T ratio of 5:1 (FIGS. 43A and 43B).

CD3 multimeric protein complex is elucidated in FIG. 44. The complexincludes a CD3ζ chain, a CD3γ chain, and two CD3ε chains. These chainsassociate with the T-cell receptor (TCR) composing of αβ chains.

CD3CAR is Used for Graft-Versus-Host Disease (GvHD).

CD3CAR is administered to a patient prior to or after a stem celltransplant. The patient is tested for elevated levels of white bloodcells.

CD3CAR is administered to a patient prior to or after a bone marrowtransplant. The patient is tested for elevated levels of white bloodcells.

CD3CAR is administered to a patient prior to or after a tissue graft.The patient is tested for elevated levels of white blood cells.

Organ Transplant

CD3CAR is administered to an organ transplant patient before organtransplant surgery. The patient is tested for organ rejection. Thefollowing histological signs are determined: (1) infiltrating T cells,in some cases accompanied by infiltrating eosinophils, plasma cells, andneutrophils, particularly in telltale ratios, (2) structural compromiseof tissue anatomy, varying by tissue type transplanted, and (3) injuryto blood vessels.

CD3CAR is administered to an organ transplant patient after organtransplant surgery. The patient is tested for organ rejection. Thefollowing histological signs are determined: (1) infiltrating T cells,in some cases accompanied by infiltrating eosinophils, plasma cells, andneutrophils, particularly in telltale ratios, (2) structural compromiseof tissue anatomy, varying by tissue type transplanted, and (3) injuryto blood vessels.

1.-105. (canceled)
 106. An engineered cell, said engineered cellcomprises: engineered chimeric antigen receptor polynucleotide, thepolynucleotide encodes for a chimeric antigen receptor polypeptidecomprising: a signal peptide, an antigen recognition domain, a hingeregion, a transmembrane domain, at least one co-stimulatory domain, anda signaling domain; and wherein the antigen recognition domain isselected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, andCD52.
 107. The engineered cell according to claim 106, wherein theantigen recognition domain is selected from the group consisting of CD2,CD3, CD4, CD5, and CD7.
 108. The engineered cell according to claim 106,wherein the engineered cell is deficient in at least one cell surfaceantigen selected from the group consisting of CD2, CD3, CD4, CD5, andCD7.
 109. The engineered cell according to claim 106, wherein theengineered cell is deficient in a CD5 cell surface antigen.
 110. Theengineered cell according to claim 106, wherein the engineered cell is aT cell, NK T cell, or NK cell.
 111. The engineered cell according toclaim 106, wherein the antigen recognition domain is CD4; and theengineered cell is NK cell.
 112. The engineered cell according to claim106, wherein the hinge region comprises a CD8 hinge region.
 113. Theengineered cell according to claim 106, wherein the transmembrane regioncomprises a CD8 transmembrane region.
 114. The engineered cell accordingto claim 106, wherein the transmembrane region comprises a CD28transmembrane region.
 115. The engineered cell according to claim 106,wherein the chimeric antigen receptor polypeptide comprises at least twoco-stimulatory domains.
 116. The engineered cell according to claim 106,wherein the chimeric antigen receptor polypeptide comprises a 4-1BB orCD28 co-stimulatory domain and a CD3zeta signaling domain.
 117. Theengineered cell according to claim 106, wherein the antigen recognitiondomain is selected from the group consisting of CD2, CD3, CD5, and CD7;and the engineered cell comprises a T cell that is deficient in a cellsurface antigen selected from the group consisting of CD2, CD3, CD5, andCD7.
 118. The engineered cell according to claim 106, wherein theantigen recognition domain is selected from the group consisting of CD2and CD7; and the engineered cell comprises an NK cell that is deficientin a cell surface antigen selected from the group consisting of CD2 andCD7.
 119. The method according to claim 106, wherein the CD2, CD3, CD5,or CD7 associated cell proliferative disease comprises precursor Tlymphoblastic leukemia/lymphoma, mature T cell lymphomas/leukemias,EBV-positive T-cell lymphoproliferative disorders, adult T-cellleukemia/lymphoma, mycosis fungoides/sezary syndrome, primary cutaneousCD30-positive T-cell lymphoproliferative disorders, peripheral T-celllymphoma (not otherwise specified), adult T cell lymphoma, T cellprolymphocytic leukemia, angioimmunoblastic T-cell lymphoma, oranaplastic large cell lymphoma.
 120. A method of generating a CD4CARengineered cells, said method comprising the steps of: obtaining apopulation of cells comprising CD4 T cells and CD8 T cells; transformingthe population of cells comprising CD4 T cells and CD8 T cells with apolynucleotide that encodes for a chimeric antigen receptor polypeptidecomprising: a signal peptide, a CD4 antigen recognition domain, a hingeregion, a transmembrane domain, at least one co-stimulatory domain, anda signaling domain; and expanding the population of cells comprising CD4T cells and CD8 T cells to provide CD4CAR engineered cells.
 121. Themethod according to claim 120, wherein the population of CD4 T cells andCD8 T cells are obtained from human cord blood.
 122. The methodaccording to claim 120, wherein the CD4CAR engineered cells are CD8 Tcells.
 123. The method according to claim 120, wherein CD4 T cells aredepleted.
 124. The method according to claim 120, wherein the CD4CARengineered cells are enriched for CD8 T cells and bear a central memoryT cell like immunophenotype.
 125. The method according to claim 120,wherein expanding comprises contacting the population of cellscomprising CD4 T cells and CD8 T cells to at least one of IL-2, IL-7,and IL-15.
 126. The method according to claim 125, wherein expandingoccurs in vivo.
 127. The method according to claim 120, furthercomprising isolating the CD4CAR engineered cells.
 128. A method oftreating a CD4 associated cell proliferative disease in a human patient,said method comprising: obtaining a population of cells comprising CD4 Tcells and CD8 T cells; transforming the population of cells comprisingCD4 T cells and CD8 T cells with a polynucleotide that encodes for achimeric antigen receptor polypeptide comprising: a signal peptide, aCD4 antigen recognition domain, a hinge region, a transmembrane domain,at least one co-stimulatory domain, and a signaling domain; expandingthe population of cells comprising CD4 T cells and CD8 T cells toprovide CD4CAR engineered cells; administering the CD4CAR engineeredcells to the human patient; and reducing the tumor burden of cellproliferative disease cells in the human patient.
 129. The methodaccording to claim 128, wherein the CD4 associated cell proliferativedisease comprises acute myeloid leukemia, acute myelomonocytic leukemia,acute monoblastic leukemia, monocytic leukemia, or chronicmyelomonocytic leukemia.
 130. The method according to claim 128, whereinthe CD4 associated cell proliferative disease comprises precursor Tlymphoblastic leukemia/lymphoma, mature T cell lymphomas/leukemias,EBV-positive T-cell lymphoproliferative disorders, adult T-cellleukemia/lymphoma, mycosis fungoides/sezary syndrome, primary cutaneousCD30-positive T-cell lymphoproliferative disorders, peripheral T-celllymphoma (not otherwise specified), adult T cell lymphoma, T cellprolymphocytic leukemia, angioimmunoblastic T-cell lymphoma, oranaplastic large cell lymphoma.
 131. The method according to claim 128,wherein the method further comprises administration in conjunction withone or more of chemotherapy, radiation, immunosuppressive agents, andantiviral therapy.
 132. The method according to claim 128, whereinexpanding comprises contacting the population of cells comprising CD4 Tcells and CD8 T cells to at least one of IL-2, IL-7, and IL-15.
 133. Themethod according to claim 132, wherein expanding occurs in vivo. 134.The method according to claim 128, wherein administering furthercomprises co-administering with at least one of cytokines, inhibitors ofcolony stimulating factor-1 receptor (CSF1R), cyclosporin, azathioprine,methotrexate, mycophenolate, CAMPATH, antibody, anti-CD3 antibody,cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, and FR901228.